Program on Negotiation – Harvard Law School – Daily Blog -> Renegotiate Salary to Your Advantage @ AT A GLANCE – Article: Generating mouse models for biomedical research: technological advances. © 2019. Channabasavaiah B. Gurumurthy, Kevin C. Kent Lloyd. Published by The Company of Biologists Ltd | Disease Models & Mechanisms (2019). Published 8 January 2019 @ Mouse Model – An Overview & Mouse models of human disease – An evolutionary perspective & Mouse model of human diseases and other very important videos about this issue @ A Handbook of Mouse Models of Cardiovascular Disease – Video & Animal models – Very Important Links @ How To Reference – Harvard Style Referencing Guide | Swinburne Online @ Harvard referencing using Microsoft Word & A guide to Harvard Referencing @ A Advanced Research Projects Agency Network (ARPANET) & Internet History Timeline: ARPANET to the World Wide Web @ How to publish a paper in Nature and other very important topics about this issue @ LINKS AND VIDEOS

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Characterization of genetic subclonal evolution in pancreatic cancer mouse models

Nature Communications volume 10, Article number: 5435 (2019)

 

Overview of Transgenic Mouse Models for Alzheimer’s Disease https://currentprotocols.onlinelibrary.wiley.com/doi/10.1002/cpns.81

Ariana MyersPaul McGonigleFirst published: 19 August 2019

Animal models in biological and biomedical research – experimental and ethical concerns http://www.scielo.br/pdf/aabc/v91s1/0001-3765-aabc-201720170238.pdf

Animal Models of Cardiovascular Diseases https://www.researchgate.net/publication/50395974_Animal_Models_of_Cardiovascular_Diseases

Heart failure and mouse models https://dmm.biologists.org/content/3/3-4/138

WEBINAR: MOUSE MODELS OF CARDIOVASCULAR DISEASE The Jackson Laboratory https://www.jax.org/education-and-learning/education-calendar/webinars/on-demand/on-demand-mouse-models-of-cardiovascular-disease

Animal models of coronary heart disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5274506/

Small mammalian animal models of heart disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5030387/

Animal models of cardiovascular diseases. https://www.ncbi.nlm.nih.gov/pubmed/21403831

Review Article

Animal Models of Cardiovascular Diseases

https://www.hindawi.com/journals/bmri/2011/497841/

Animal Models of Cardiovascular Disease

Modelos animales de enfermedad cardiovascular

https://www.revespcardiol.org/en-animal-models-cardiovascular-disease-articulo-13131649

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4875775/

https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/mouse-model

https://dmm.biologists.org/content/12/1/dmm029462

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Renegotiate Salary to Your Advantage

When it’s time to renegotiate salary, we often downplay our contributions to the organization. Here’s advice on how to bargain up rather than down—and earn what you truly deserve.

BY KATIE SHONK — ON DECEMBER 30TH, 2019 / SALARY NEGOTIATIONS

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renegotiate salary

As we prepare to renegotiate salary, most of us intend to ask for as much as we can without antagonizing our employer. But we sometimes undervalue our worth, with disappointing consequences.

To take one dramatic example, in 2013, the board of Chicago public radio station WBEZ offered a big raise—more than $100,000—to Ira Glass, the creator and host of the long-running radio show This American Life, to recognize his value to the organization. Glass’s salary would go from $170,000 to $278,000.

But the big bump in pay made Glass uncomfortable, he told the New York Times, so he asked the board to lower his salary to $146,000–less than the starting point of the negotiation. Later, he reportedly asked for his salary to be lowered again, even though he was having to book speaking engagements to cover his and his wife’s living expenses in New York City.

Discover how to refine your negotiation skills with this free special report, Salary Negotiations: How to Negotiate Salary: Learn the Best Techniques to Help You Manage the Most Difficult Salary Negotiations and What You Need to Know When Asking for a Raise, from Harvard Law School.

Why would Glass negotiate his salary down rather than up? He may have felt self-conscious about earning a high salary from a not-for-profit organization funded by grants and listener donations.

Negotiation is difficult enough without creating extra roadblocks for ourselves, yet that’s what we often do. This tendency can be particularly strong when we try to renegotiate salary, as we often feel vulnerable and insecure about our worth. Our salary negotiation tips will help ensure you don’t sell yourself short.

Get Out of Your Way

To renegotiate salary and other job terms more effectively, we have to recognize how we “get in our own way,” write Simmons School of Management professor emerita Deborah M. Kolb and Jessica L. Porter in their book Negotiating at Work: Turn Small Wins Into Big Gains (Jossey-Bass, 2015). Pitfalls include failing to recognize opportunities to negotiate, focusing on our own weaknesses, and making the first concessions in our own heads, before other parties have voiced their positions.

Bargaining ourselves down starts with self-doubt about our value. Before we renegotiate salary, we tend to think that the employer has all the cards—that our only choices are to acquiesce or reject an offer outright. These internal dialogues are where the first concessions in the negotiation are made, write Kolb and Porter. We might decide not to renegotiate salary because we want to negotiate hard on another issue, rather than looking for ways to negotiate across multiple issues.

When we fail to recognize our own value, we are vulnerable to accepting less than we’re entitled to. In addition, our beliefs about what will satisfy the other party may be incorrect. Glass’s employer, for example, might have wanted him to accept a raise that would enable him to focus fully on his work without the need to overtax himself with side jobs.

How to Bargain Salary

When preparing to renegotiate salary, there are a number of steps you should take to be an effective self-advocate, according to Kolb and Porter:

  1. Gather information so that you will feel that what you are asking for is defensible. Prepare to explain the value you bring to the organization.
  2. Develop alternatives to the current negotiation to increase your flexibility at the table. Keep in mind that the other party’s alternatives, such as losing you, may be less attractive than yours.
  3. Examine your vulnerabilities and plan ahead to compensate for them. For example, if a project you worked on didn’t pan out, prepare to discuss what went wrong, what went right, and how you learned from the situation.

A Word about Anchors

Abundant negotiation research has found that whatever figure is introduced first into a negotiation—however arbitrary or unfair it may be—serves as a powerful anchor that pulls the discussion it its direction. When we renegotiate salary, what’s the most obvious anchor? Your current salary. Like it or not, whatever you earn now will anchor the salary discussion.

Fortunately, when asking for a raise, you may be able to identify another anchor that would be more advantageous to you. For example, suppose you believe you are significantly underpaid. Research what you believe you should be making and secure documentation, such as job postings and information from industry sources. Present this data when you meet to renegotiate salary. Although your current salary will remain salient in your boss’s mind, you might be able to lessen its impact with a new anchor.

What other business negotiation solutions have you found helpful when trying to renegotiate salary?

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Animal Models of Cardiovascular Diseases

Article· Literature Review (PDF Available)inJournal of Biomedicine and Biotechnology 2011(2):497841 · February 2011 with 1,091 Reads DOI: 10.1155/2011/497841 · Source: PubMedCite this publication

Show more authorsAbstractCardiovascular diseases are the first leading cause of death and morbidity in developed countries. The use of animal models have contributed to increase our knowledge, providing new approaches focused to improve the diagnostic and the treatment of these pathologies. Several models have been developed to address cardiovascular complications, including atherothrombotic and cardiac diseases, and the same pathology have been successfully recreated in different species, including small and big animal models of disease. However, genetic and environmental factors play a significant role in cardiovascular pathophysiology, making difficult to match a particular disease, with a single experimental model. Therefore, no exclusive method perfectly recreates the human complication, and depending on the model, additional considerations of cost, infrastructure, and the requirement for specialized personnel, should also have in mind. Considering all these facts, and depending on the budgets available, models should be selected that best reproduce the disease being investigated. Here we will describe models of atherothrombotic diseases, including expanding and occlusive animal models, as well as models of heart failure. Given the wide range of models available, today it is possible to devise the best strategy, which may help us to find more efficient and reliable solutions against human cardiovascular diseases.

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Figures – uploaded by Carlos TarínAuthor contentContent may be subject to copyright.: Animal models of atherosclerosis: advantages and limitations.… Content uploaded by Carlos TarínAuthor contentContent may be subject to copyright.Download full-text PDFOther full-text sourcesHindawi Publishing CorporationJournal of Biomedicine and BiotechnologyVolume 2011, Article ID 497841, 13 pagesdoi:10.1155/2011/497841Review ArticleAnimal Models of Cardiovascular DiseasesCarlos Zaragoza,1Carmen Gomez-Guerrero,2Jose Luis Martin-Ventura,2Luis Blanco-Colio,2Bego˜na Lavin,1Be˜nat Mallavia,2Carlos Tarin,2Sebastian Mas,2Alberto Ortiz,2and Jesus Egido21Department of Epidemiology, Atherothrombosis and Cardiovascular Imaging, Fundacion Centro Nacional InvestigacionesCardiovasculares Carlos III (CNIC), Sinesio Delgado 3, 28029 Madrid, Spain2Renal and Vascular Research Laboratory, IIS-Fundacion Jimenez Diaz, Universidad Autonoma, Avda Reyes Catolicos 2,28040 Madrid, SpainCorrespondence should be addressed to Jesus Egido, jegido@fjd.esReceived 11 October 2010; Revised 4 January 2011; Accepted 17 January 2011Academic Editor: Oreste GualilloCopyright © 2011 Carlos Zaragoza et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.Cardiovascular diseases are the first leading cause of death and morbidity in developed countries. The use of animal models havecontributed to increase our knowledge, providing new approaches focused to improve the diagnostic and the treatment of thesepathologies. Several models have been developed to address cardiovascular complications, including atherothrombotic and cardiacdiseases, and the same pathology have been successfully recreated in different species, including small and big animal models ofdisease. However, genetic and environmental factors play a significant role in cardiovascular pathophysiology, making difficultto match a particular disease, with a single experimental model. Therefore, no exclusive method perfectly recreates the humancomplication, and depending on the model, additional considerations of cost, infrastructure, and the requirement for specializedpersonnel, should also have in mind. Considering all these facts, and depending on the budgets available, models should be selectedthat best reproduce the disease being investigated. Here we will describe models of atherothrombotic diseases, including expandingand occlusive animal models, as well as models of heart failure. Given the wide range of models available, today it is possible todevise the best strategy, which may help us to find more efficient and reliable solutions against human cardiovascular diseases.1. IntroductionCardiovascular diseases are the first leading cause of deathand morbidity in developed countries. Cardiac and vascularcomplications are complex multifactorial pathologies, inwhich both genetic and environmental factors are impli-cated, thus making them very difficult to prevent. Thedevelopment of animal models of cardiovascular disease(CVD), including cardiac and atherothrombotic diseases,has provided us today with important insights into thepathophysiology, and they were found to be essential tools toevaluate new therapeutic strategies to predict and to preventthese complications.Here, we will summarize the most common modelsof cardiovascular diseases, including those implemented inboth large and small animals, designed for helping to coverwith more precision and to better understand every singleaspect related to these human pathologies. In particular, wewill describe models of atherothrombotic diseases, includingexpanding abdominal aortic aneurysms (AAA), thoracicaneurysms, and occlusive atherosclerotic diseases, as well asmodels of heart failure. These situations constitute todaya significant challenge since predictors to evaluate earlydetection and forecast progression are crucial in thesepathologies, yet they are poorly explored.2. Animal Models of Atherothrombotic Disease2.1. Mouse Models. Atherosclerosis is a complex multifac-torial disease with different etiologies that synergisticallypromote lesion development. Mouse models have provedto be useful to study development and progression ofatherosclerotic lesion, and several reviews have extensivelydiscussed the different available models [1–3]. In particular,2Journal of Biomedicine and Biotechnologyknockout and transgenic mouse models for atherosclerosishave been instrumental in understanding the molecular andcellular mechanisms involved in atherogenesis, and in eval-uating the effectiveness of new and existing atheroscleroticdrugs [4].As wild-type mice are resistant to lesion develop-ment, the current mouse models for atherosclerosis arebased on genetic modifications of lipoprotein metabolismwith additional dietary changes. Among them, low-densitylipoprotein receptor-deficient mice (LDLR−/−mice) andapolipoprotein E-deficient mice (apoE−/−mice) are themost widely used. Atherosclerotic lesions seen in thesemodels can be exacerbated by the addition of risk factorssuch as hypertension or diabetes. Mice have become widelyused as models of human atherosclerosis as they offeradvantage compared with other species (Ta bl e 1 ).2.2. LDLR−/−Mice. The LDLR−/−mouse represents amodel of familial hypercholesterolemia due to one of themutations affecting the LDLR, and the plasma lipoproteinprofile resembles that of humans. Mice, which are genet-ically deficient in LDLR manifested delayed clearance ofVLDL and LDL from plasma. As a result, LDLR−/−miceexhibit a moderate increase of plasma cholesterol level anddevelop atherosclerosis slowly on normal chow diet [5,6].Interestingly, the severity of the hypercholesterolemia andatherosclerotic lesions in LDLR−/−mice can be acceleratedby feeding a high-fat, high-cholesterol diet [5–7], by mutat-ing the apoB gene into an uneditable version [8,9], andby crossing either with leptin-deficient mice [10]orwithapoB100 transgenic mice [11]. Under these conditions, thelesions in the aorta can progress beyond the foam-cell fatty-streak stage to the fibroproliferative intermediate stage.In addition to LDLR−/−mice, the LDLR and apoEdouble-deficient mouse (LDLR−/−apoE−/−)whichdevel-ops severe hyperlipidemia and atherosclerosis even ona regular chow diet, has been proposed as a suitablemodel to study the antiatherosclerotic effect of compoundswithout having to feed the animals an atherogenic diet[12,13]. However, the response of both LDLR−/−andLDLR−/−apoE−/−mice to the treatment with hypolipi-demic drugs varies from lowering of plasma cholesterolwithout atherosclerosis decrease to a weak lesion reductionwith or without lower plasma cholesterol [4,14]. By contrast,those mice effectively respond to agonists of peroxisomeproliferator-activated receptor (PPAR) or liver X receptor[15,16]. This great variability indicates that LDLR−/−is probably not well-suited for analyzing the cholesterol-lowering and antiatherogenic effects of drugs.2.3. ApoE−/−Mice. In 1992 two different groups simultane-ously generated the apoE−/−mice by homologous recom-bination in embryonic stem cells [17,18]. Homozygousdeficiency in apoE gene results in a marked increase in theplasma levels of LDL and VLDL due to a failure in theirclearance through the LDLR and LDLR-related proteins. TheapoE−/−mouse contains the entire spectrum of lesionsobserved during atherogenesis and was the first mouse modeldescribed to develop lesions similar to those of human [17,18].Under normal dietary conditions, apoE−/−mice havedramatically elevated plasma levels of cholesterol, and theydevelop extensive atherosclerotic lesions widely distributedthroughout the aorta [18–20]. This process can be exacer-batedonahigh-fatdiet,withfemalemicemoresusceptiblethan male mice [19]. A chronological analysis of atheroscle-rotic lesions in apoE−/−mice revealed that the sequentialevents involved in lesion formation in this model are strik-ingly similar to those in larger animal models and in humans.Predilection sites for atherosclerotic lesions in apoE−/−mice are the aortic root, followed by the aortic arch, thebrachiocephalic trunk, the left carotid, and subclavian andcoronary arteries [6,21]. Aortic lesions rapidly develop frominitial fatty streaks comprised primarily of foam cells withmigrating smooth muscle cells to more complex lesions inmiddle-aged mice. These advanced lesions are heterogeneousbut typically composed of a necrotic core surrounded byproliferating smooth muscle cells and extracellular matrixproteins [20,22].The apoE−/−mice are currently the most widely utilizedanimal model for the study of atherosclerosis. In fact, theeffect of many genes on the development of atherosclerosishas been examined by crossing the apoE−/−mice with othergenetically manipulated animals. Furthermore, the apoE−/−mouse serves as a useful tool to: (i) identify atherosclerosis-susceptibility-modifying genes, by the candidate-gene andgene-mapping methods, (ii) decipher molecular mechanismand cell types involved in atherogenesis, (iii) search intothe drug effects on atherosclerosis, and (iv) assess noveltherapies that prevent lesion progression. In this sense,the apoE−/−mouse model was used to test additionaltherapeutic effects of statins beyond those attributablesolely to cholesterollowering. One of the first observationswas the paradoxical effect of simvastatin on atherogenesisin both apoE−/−and LDLR−/−mice [23]. In contrastto the atheroprotective effect of simvastatin in LDLR−/−mice, age-matched apoE−/−showed elevated serum totalcholesterol and increased aortic plaque area, thus suggestingthat the therapeutic effect of simvastatin may depend onthepresenceofafunctionalapoE[23]. In spite of this,the antiatherosclerotic effects of other statins have beeneffectively proven in apoE−/−mice [24,25]. Several othercompounds, such as angiotensin II receptor antagonists orPPAR agonists [26] also reduced the extent and severity ofatherosclerotic lesions without lowering plasma cholesterolin apoE−/−mice.However,therecentfindingofincreasedatherogenesis in apoE−/−mice treated with PPAR alpha andPPAR gamma agonists is consistent with clinical findings ofthe adverse cardiovascular events of dual therapy [27].Nevertheless, a major limitation of the apoE−/−mousemodel is the infrequency of plaque rupture and thrombo-sis, two common complications of human atherosclerosis.Ischemic cardiomyopathy has been occasionally found inaged mice [20], but interestingly, rapid coronary arteryocclusion, myocardial infarction, and even premature deathoccur when apoE−/−mice were crossed with mice deficientin scavenger receptor class B type I or its adaptor proteinJournal of Biomedicine and Biotechnology 3Tab le 1: Animal models of atherosclerosis: advantages and limitations.Mouse(i) Rapid development of atherosclerotic plaques (i) Only partial resemblance to humans(ii) Short reproductive cycle (ii) More atherosclerotic than atherothrombosis model(iii) Large litters (iii) Very high levels of blood lipids(iv) Well-known genome(v) Relative ease of genome manipulation(vi) Relatively cheap(vii) Useful for noninvasive imaging(MRI,PET,CT,ultrasound)(viii) Large experienceRat(i) Easy, available, and cheap (i) Do not develop atheroma(ii) Useful for restenosis analysisRabbit(i) Medium size (i) Need for high blood cholesterol levels(ii) Fibroatheroma lesions (ii) No plaque rupture model(iii) Useful for restenosis models (iii) A model of neointima formation rather thanatherosclerosis(iv) AffordablePorcine(i) Lesions more similar to human disease (i) High cost(ii) Valid for restenosis studies (ii) Difficult handling(iii) Few genomic tools[28,29], thus mimicking many cardinal features of humancoronary heart disease.2.4. Transgenic Mice. Transgenic technologies have provideda series of very useful mouse models to study hyperlipi-demia and atherosclerosis. Among them, mice expressingmutant forms of apoE, such as apoE3Leiden (E3L) andapoE (Arg 112 →Cys →142) transgenic mice, are the morewidely studied. These mice display a lipoprotein profilecomparable to that of patients with dysbetalipoproteinemia,in which plasma total cholesterol and triglycerides are mainlyconfined to (V) LDL [30]. The E3L transgenic mice developatherosclerotic lesions with all the characteristics of humanvasculopathy, varying from fatty streak to mild, moderate,and severe plaques [30,31]. Furthermore, E3L transgenicmice and the more recently developed E3L/Cholesteryl estertransfer protein (CETP) transgenic mice have been shownto be more sensitive to a variety of hypolipidemic drugs andPPAR agonists than apoE−/−and LDLR−/−mice [4,32].2.5. Mouse Models of Diabetes-Accelerated Atherosclerosis.Diabetes is a high risk factor of cardiovascular disease.The cardiovascular complications of diabetes are manifestedprimarily as ischemic heart disease caused by acceleratedatherosclerosis, and also as cardiomyopathy. Several modelsare available to study atherosclerosis and cardiomyopathyassociated with diabetes, including apoE−/−and LDLR −/−mice in which type 1 diabetes is induced by streptozotocinor viral injection [33,34]. In both mice, diabetes induc-tion did not markedly change plasma lipid levels, therebymimicking the accelerated atherosclerosis seen in patientswith type 1 diabetes. Importantly, streptozotocin-injectedatherosclerotic mice exhibited increased atherosclerosis inthe aortic sinus, carotid artery, and abdominal aorta, as wellas calcifications in the proximal aorta [34,35].In brief, mouse models have been very useful to unveilthe importance of inflammatory and immunological mecha-nisms in the formation and progression of atheroma plaque.Recently, an enormous interest for the use of noninvasivemagnetic resonance imaging (MRI) in mouse models ofatherosclerosis has arisen [36], since MRI accurately char-acterizes the location, the size and the shape of lesions.In addition, MRI allows the differentiation between fibrousand lipid components of regress in plaques in mice. Incombination with noninvasive imaging technologies, mousemodels of atherosclerosis today also serve to test novel4Journal of Biomedicine and Biotechnologycontrast agents, and to design and target specific moleculesinvolved in high-risk plaque.2.6. Rabbit Models. The high-cholesterol diet rabbit modelhas been widely used for experimental atherosclerosis. Backin 1913, cholesterol was found to cause atheroscleroticchanges in the rabbit arterial intima, which is very similar tohuman atherosclerosis. Atherosclerotic lesions also developin normolipemic rabbits as a result of repeated, or con-tinuous intimal injury by an indwelling aortic polyethylenecatheter, balloon angioplasty or nitrogen exposure. There-fore, many studies have used the rabbit model with high-cholesterol diet, arterial wall injury, or, most commonly, acombination of these two methods. In all these models, theobserved lesions resemble, at least in part, those seen inhuman plaques, mainly regarding the inflammatory compo-nent, though the vascular smooth muscle cell proliferationdetermines for a great deal the lesion.The rabbit model has largely been used to study theinfluence of lipid lowering (by diet or statins) on theplaque formation and “stabilization.” Those studies havecontributed to unveil the mechanisms by which lipid lower-ing reduces macrophage accumulation and other aspects ofatheroma inflammation [37,38].Recently, we have set up a novel rabbit model to examinethe influence of inflammation on atherosclerotic plaque. Theaim was to study some mechanisms by which atherosclerosisis particularly severe in patients with rheumatoid arthritis.Briefly, the model consists in a combination of femoral injuryin hyperlipidemic rabbits and induced acute knee arthritis.Those animals had more intensive vascular lesions thananimals without inflammation. This model could representa novel approach to the study, inflammation-associatedatherosclerosis [39].A model for plaque rupture has been also developedin rabbits. Shimizu and coworkers [40] have developed asimple rabbit model of vulnerable atherosclerotic plaque,with the combination of aggressive vascular injury associatedto a hyperlipidemic diet. The histological findings showedthat an aortic plaque had the three features of “vulnerableplaque”: lipid-rich core, accumulation of macrophages, anda thin fibrous cap. In addition, a low-density lipoprotein(LDL) receptor-deficient animal model (the WHHL rabbit)has been developed. This model resembles to human familialhypercholesterolemia and shows evidence of progressivedisease of the aorta with accumulation of birefringent lipidsin intimal lesions and plaques, as well as in the media frombirth to 1 year of age.Although rabbit aortic arteries are smaller in vesseldiameter than human carotid artery, they allows the studieswith endovascular therapeutic devices. In addition, therabbit model has also been used for the quantificationof atherosclerotic aortic component by MRI. This tech-nique accurately quantifies fibrotic and lipid componentsof atherosclerosis in the model and may permit the serialanalysis of therapeutic strategies on atherosclerotic plaquestabilization [41].2.7. Porcine Models. They prevention of heart attack andstroke depends on the detection of vulnerable plaquesand development of plaque-stabilizing therapies. Animalmodels are essential for testing mechanistic hypotheses ina controlled manner, they should be representative of ahuman disease, and at the same time be easy to manip-ulate. However, vulnerable plaque recreation is one of thetoughest tasks in animal model design. Plaque rupture isan additional complication of an already complex process,and the precise mechanisms involved remain hypothetical.A plethora of experimental approaches are available forgrowing atherosclerotic lesions in various animal species asmentioned above (Ta b l e 2 ).Currently, there is no single and golden standard animalmodel of vulnerable plaque, but pig models are probably thebest way to recreate human plaque instability. The combina-tion of diabetes and hypercholesterolemia constitutes a goodmodel of accelerated atherosclerosis [42], and it was relevantstudy the role of certain biomarkers, such as the Lp-PLA2since these animals share a similar plasma lipoprotein profilehumans. In this regard, the selective inhibition of Lp-PLA2by darapladib decreased progression to advanced coronaryatherosclerotic lesions and confirmed a crucial role of vascu-lar inflammation not associated to hypercholesterolemia, inthe development of lesions implicated in the pathogenesis ofmyocardial infarction and stroke [43].Several porcine models of advanced human-like coro-nary atherosclerosis have been employed to analyze thedevelopment and validation of coronary imaging technolo-gies. In the evolving era of technological development, theavailability and use of such animal models will becomecritical for the development of emerging technologies ininterventional cardiology [44], and for the study of drug-eluting stents [45]. In addition, the porcine models ofcoronary atherosclerosis allow examining the impact ofadventitial neovascularisation, on atherosclerotic plaquecomposition and vascular remodelling [46].3. Animal Models of Abdominal AorticAneurysms (AAAs)Animal models of atherothrombotic AAA are essential toolsfor the preclinical evaluation of new therapeutic strategiesfor the suppression of aneurysmal degeneration (Ta bl e 3 ).Recent insights into the mechanisms of human AAA havecome from the studies in mouse models, and elastase-induced AAA in particular appears to recapitulate manyfeatures of human AAA. Here we briefly outline the mostfrequently used models of AAAs, and refer the reader torecent comprehensive reviews regarding additional animalmodels [47–52].3.1. Rat Models3.1.1. Localized Aortic Perfusion with Elastase. This modelconsists of exposing a segment of the abdominal aorta andinfusing it with elastase [53]. The degradation of elasticfibers triggers an inflammatory response that develops intoJournal of Biomedicine and Biotechnology 5Tab le 2: Animal models of plaque rupture and plaque associated thrombosis.Spontaneous Induced39–54-month-old pigs with inherited hyper=LDLcholesterolemia. ApoE−/−mice after squeezing the aorta supplemented between forceps.42–54-week old ApoE−/−mice. Hypercholesterolemic rabbits subjected to balloon injury.Dahl salt-sensitive hypertensive transgenic rats for humancholesteryl ester transfer protein. Atherosclerotic ApoE−/−mice subjected to photochemical injury.—Intraperitoneal injection of Russell’s viper venom in New Zealand Whiterabbits intermittently fed with high cholesterol diet.Intraperitoneal injection of Russel’s viper venom in Watanabe heritablehyperlipidemic rabbits, in combination with the administration ofserotonin or angiotensin II.an aneurysm [54,55]. The severity of the induced AAA canbe increased by adding plasmin to the infusion. This modelhas been adapted for use in several other species, includingrabbit, mouse, and large animals.3.1.2. Decellularized Xenografts. This model was based onthe observation that chronic rejection of arterial allograftsand xenografts, results in arterial wall dilatation and rupture.Michel and coworkers decellularized a section of abdominalaorta from one species (e.g., guinea pig), and the resultingtube of intact extracellular matrix was grafted into anothermorphologically compatible species, usually rat [58]. Thexenogenic extracellular matrix triggers the destruction ofhost matrix, leading to aneurysm formation. The model hasbeen successfully used to evaluate therapeutic targets [64–69], although the heterogeneity of the aneurysms formed andthe lack of vessel rupture are significant limitations.3.2. Mouse Models. The mouse has become the preferredmodel for cardiovascular research for several reasons, includ-ing the ease of handling, low procedure costs, and theability to manipulate the mouse genome. Consequently, of allanimal models of AAA, mouse models have provided mostof the insights into the mechanisms of human AAA. Thefollowing models are the most widely-used to date.3.2.1. Calcium-Chloride-Induced AAA. In this method, ini-tially developed in rabbits [62], calcium chloride is appliedperiaortically in the region between the renal arteries andthe iliac bifurcation. Significant dilatation of the aorta isobserved within 14 days, and the severity is significantlyincreased if calcium chloride is applied together with thio-glycolate and if animals are fed a high-cholesterol diet [56].Unlike other models, calcium chloride application inducesAAA without the need for mechanical intervention.3.2.2. Elastase-Induced AAA. The elastase-induced modelwas adapted for mice by Pyo et al. [57]. Elastase perfusion inmouse aorta causes a mild-to-moderate dilatation initially,which subsequently develops to a >100% increase in aorticdiameter within 14 days. In this model, the degradationof medial elastin is delayed, and the subsequent aorticwall inflammation consists of mononuclear phagocytesthroughout the adventitial and medial layers, with relativelyfew polymorphonuclear cells localized to the adventitia[57]. Elastase-induced injury increases the expression ofMMPs, cathepsins, and other proteases [70], with MMP-9being localized to aneurysm-infiltrating macrophages [71].Elastase-induced AAAs thus appear to recapitulate manyfeatures of human AAAs, and this model has become avaluable and convenient tool for systematically evaluating theroles of individual gene products in aneurysmal degeneration[71–80].When compared to calcium-chloride-induced AAA, themain limitation of this method is in the mechanical stressrequired to recreate medial elastic degradation. However,the protocol resembles the time course of events leadingto human AAA, including initial recruitment of leukocytesand mast cells, the development of a transmural aorticwall inflammatory response, and finally the upregulation ofextracellular matrix metalloproteinases and other proteases,which induce a progressive degradation of the medial elastinand collagen, leading to the final aortic dilatation.3.2.3. Angiotensin II-Induced AAA. This procedure was ini-tially developed to define whether increased plasma concen-trations of Angiotensin II (Ang II), have a direct effect on theatherogenic process in hyperlipidemic old apoE−/−mice.Unexpectedly, Ang II also produced large suprarenal abdom-inal aortic aneurysms in these animals [81]. In this model,inflammation of the vessel wall is associated with signalingthrough AT1a receptors [82], nuclear factor- (NF-) kappaB-mediated induction of proinflammatory genes, includingMCP-1, M-CSF, iNOS, COX-2, inhibition of PPARs [83],activation of the NADPH oxidase p47phox [84], c-JUN N-terminal [85], Rho kinases [86], and enhanced recruitmentof macrophages [87,88] and extracellular matrix compo-nents and degrading enzymes [89–91], leading to vesseldissection, and rupture. The severity of AAA is higher inhyperlipidemic apoE−/−or LDLR−/−male mice (∼60%of mice), when compared to normolipidemic C57Bl/6 mice,although in these models neither hyperlipidemia per se noratherosclerosis is considered major determinants [92–94].The model contributed to evidence the implication ofthe rennin-angiotensin (RAS) system in aneurysmal disease.However, two main limitations should be considered: the6Journal of Biomedicine and BiotechnologyTab le 3: Current procedures for inducing AAA in animals.Species AAA modelsMurineCalcium Chloride [56].Elastase infusion [57].Angiotensin-II-infused AAA: used in hyperlipidemic (ApoE−/−or LDLR−/−) male mice or in wild-type C57BL/6 mice inconjunction with repeated administration of neutralizing TGF-βantibodies [50].Decellularized xenografts: grafting of abdominal aortic extracellular matrix from one species to a compatible recipient of adifferent species [58].Spontaneous [59].Rabbit Elastase-induced AAA: similar to the murine model [60], also applicable to the carotid artery [61].Calcium chloride [62].Pig Surgical model: dilatation of the infrarenal aorta with an angioplasty balloon followed by infusion of pancreatic porcineelastase [63].suprarenal location of the aneurysm (in contrast to theinfrarenal location in humans) and the clinical relevanceof RAS inhibition, since the association of RAS in humanAAA has provided controversial results, pointing towardsnecessary large population studies.3.2.4. Spontaneous Mouse Mutants. The blotchy mouse isa mouse strain containing a spontaneous mutation on theX chromosome which leads to abnormal intestinal copperabsorption. These animals have weak elastic tissue due tofailed crosslinking of elastin and collagen, and develop aorticaneurysms mainly in the aortic arch, thoracic aorta, andoccasionally in the abdominal aorta [59]. However, resultsfrom this model are difficult to interpret, since the mutationproduces many severe additional effects.3.3. Rabbit Models. Several of the same interventions used inmice are also implemented in rabbits, including elastase infu-sion [60] and calcium chloride application to the abdominalaorta [56]. Another intervention used in rabbits is elastaseinfusion in the right carotid artery [61]. The main advantageof rabbits over other animal models is that rabbit aneurysmsmore closely resemble human aneurysms hemodynamicallyand histologically. Rabbit models also combine several ofthe attractive features of small animals, such as the easyhousing and handling. In addition, similarly to large animals,rabbit aneurysms can be monitored by accessing through thefemoral artery, thus providing an excellent model for testingendovascular therapies [95,96].3.4. Porcine Models. Porcine models of AAA have providedsignificant information about the changes that occur afterAAA induction and about the responses to stent deployment.A recently-developed porcine model combines mechanicaldilatation by balloon angioplasty with enzymatic degrada-tion by infusion of a collagenase/elastase solution.The modelis characterized by gradual AAA expansion associated withdegradation of aortic wall elastic fibers, an inflammatorycell infiltrate, and persistent smooth muscle cell loss [63].A broad number of similarities were found between thismodel and human AAA, and the procedure may alsorepresent an excellent method to evaluate endovascularrelated procedures. Despite the benefits, however, pigs havesignificant disadvantages, including complex animal han-dling, the requirement of special housing and surgical roomfacilities, the elevated cost of the animals, and the reducedsample sizes per assay.3.5. Thoracic Aortic Aneurysm (TAA). Elastic tissue degra-dation is also related to the development of thoracic aorticaneurysm (TAA), and mouse models have significantlyadvanced the understanding of this pathology. TAA is acharacteristic feature of Marfan syndrome (MFS), a disordercaused by mutations that affect the structure or expressionof the extracellular matrix protein fibrillin-1, a glycoproteinthat is associated of extracellular proteins, including integrinreceptors and insoluble elastin [97]. Fibrillin-1 mutationsin MFS decrease ECM sequestration of latent TGFβ,thusrendering it more prone to or accessible for activation [98–100]. TAA progression in MFS is driven by elastic fiber cal-cification, vascular wall inflammation, intimal hyperplasia,structural collapse of the vessel wall, impaired activation ofMAP kinase signaling, and altered synthesis of ECM proteinsand matrix-degrading enzymes (MMPs) [101]. Systemicadministration with TGFβantagonists has been successfullyused to mitigate vascular disease in mouse models of MFSandinchildrenwithsevereandrapidlyprogressiveMFS[97,102]. Moreover, studies in mouse models have shown thatfibulin-4 and LRP1 are also associated with TAA [103,104].4. Animal Models of Heart FailureModels of heart disease in small animals, particularly rats,have been very useful for the assessment of pharmacolog-ical therapies. In addition, several target genes have beenidentified in genetically modified mouse models. Many ofthese genes have proved to be crucial in the initiation andprogression of heart disease. Below, we describe the animalmodels currently used to study heart failure, which are alsosummarized in Ta b le 4 .4.1. Rat Models. Rat models have dominated research intoheart damage because, while rats share many of the benefitsof mice (low cost, ease of handling, etc.), their largerJournal of Biomedicine and Biotechnology 7Tab le 4: Current procedures for inducing heart damage in animals.Species Heart failure modelsMurineMyocardial damageChemical: isoproterenol administration [106].Electrical: overlapping burns [107].Surgical: coronary artery ligation [105].Ischemia Reperfusion:Transient occlusion of the left coronary artery [108].Cryoinfarction: cryo-injuries to the epicardium [109].Pressure overload: aortic constriction and banding. Aortic stenosis [110,111].Transgenic models of dilated cardiomyopathy: mutation of cardiac α-actin protein [112].RabbitSpontaneous: WHHLMI rabbits [113].Coronary artery occlusion: similar to the murine model, also applicable to the carotid artery. This is an excellentmodel for testing endovascular therapies [114].Pressure overload: aortic banding [115], valvular stenosis [116].DogMicroembolization [117].Tachycardia: ventricular pacing [118].Aortic stenosis [119].Pig Surgical model: balloon occlusion of coronary arteries [120].Tachycardia: pacing-induced supraventricular tachycardia [121].size greatly facilitates surgical and postsurgical procedures.Myocardial damage in rat hearts is induced by three proce-dures: surgical, pharmacological, or electrical.The surgical method, first developed by Pfeffer andcoworkers, consists of ligating the left coronary artery [105].In this procedure, left thoracotomy is performed on theanesthetized rat, and the heart is rapidly exteriorized bygentle pressure on the right side of the thorax. The leftcoronary artery is either ligated or heat cauterized betweenthe pulmonary artery outflow tract and the left atrium.The heart is then returned to its normal position and thethorax immediately closed. Several modifications have beenintroduced to improve performance and to reduce animalmortality, and left coronary artery ligation is the mostcommon method used to induce acute myocardial damage inrat and other animal models. One important modification istemporary occlusion followed by reperfusion, allowing flowrecovery through the previously occluded coronary arterybed. Left coronary artery ligation can thus be used toevaluatediverse parameters resulting from either permanent ischemiaor ischemia/reperfusion.Pharmacological induction of heart damage was firstimplemented by Bagdon and coworkers in 1963 and isachieved by treatment with the beta-one adrenergic receptor(B-AR) agonist isoproterenol [106]. Isoproterenol adminis-tration before ischemia exerts a cardioprotective action inrats, but at the right dose it induces cardiac myocyte necrosisand extensive LV dilatation and hypertrophy.Isoproterenol treatment and left coronary artery ligationin rats are efficient and reproducible methods that providevaluable information about the underlying mechanismsimplicated in human heart disease.The electrical method consists of generating overlappingburns in exposed rat hearts by applying a 2-mm-tippedsoldering iron to the epicardium of the left ventricle [107].While this is also a valid method, the degree of heart damageproduced is not consistent among laboratories, limiting thereproducibility of the results obtained with this procedure.4.2. Mouse Models. Against the many general advantages ofworking with mice (ease of handling, low pregnancy times,etc.), investigators choosing them as models of heart failuremust consider two important limitations: the small size ofthe heart and the structural differences with respect to thehuman cardiovascular system. Nonetheless, the availabilityof transgenic and knockout strains and the relative ease withwhich new genetic modifications can be introduced make themouse one of the most attractive models for research into themolecular basis of heart failure.One of the most widely used models of heart failure inmice is the left coronary artery ligation procedure, adaptedfrom rat. In some protocols the artery is occluded perma-nently, but recently procedures for temporary occlusion havebeen introduced to reproduce human ischemia/reperfusioninjury [108]. In this method the left anterior descendingcoronary artery is occluded and then reperfused, allowingflow recovery through the previously occluded coronaryartery bed. Reperfusion is monitored visually, and the infarctcan be analyzed by histopathological techniques, and can bedocumented in real time by non invasive high frequency.The areas at risk and the infarct size are revealed by stainingwith Evans blue dye and triphenyltetrazolium chloride andare assessed by computerized planimetry. This model hasconfirmed the benefits of reperfusion, since infarct sizewas found to be significantly lower than after permanentocclusion of the coronary artery.The method has been further modified to analyzeischemic preconditioning of the heart. In this method, theleft coronary artery is repeatedly occluded to subject theheart to several rounds of brief ischemia and reperfusion,8Journal of Biomedicine and Biotechnologyfollowed by permanent occlusion. This approach has iden-tified several ischemia-induced genes that confer tolerance toa subsequent ischemic event [122].More recently, a model of myocardial infarction wasdeveloped, in mice and rats, in which a series of cryo-injuries is generated in the heart. This new model is yieldingpromising results [109].4.3. Large Animal Models of Heart Failure. Small animalmodels have provided significant insights into human car-diac pathophysiology. However, rodent and human heartsdiffer in their architecture, heart rates, oxygen consumption,contractility, protein expression, and even stem cell popula-tions, and there is therefore an obvious need for models ofheart failure in large animals.The first large animals used to study heart failure weredogs, in which models of myocardial infarction and serialmicroembolization of the coronary artery were developed[117]. However, the preferred large animal model of heartdamage is the pig, because the collateral coronary circulationand arterial anatomy of pigs and humans are very similarand infarct size can be accurately predicted [123]. Amongseveral models of MI in pigs, one of the most widelyused is balloon occlusion of the left anterior descendingcoronary artery. In this model, a catheter is inserted throughthe femoral artery, positioning an angioplasty balloon overa guide wire at a position distal to the second largestdiagonal branch of the artery, and infarction is inducedby balloon inflation [120]. The similar size and cardiacphysiology of pigs and humans mean that this model offersmajoradvantagesovermodelsinotherspecies.However,themethod requires specialized equipment, dedicated surgicalfacilities and skilled personnel, limiting the number oflaboratories able to conduct these studies.Therabbit,muchlessexpensivethanpig,offers acompromise solution. Rabbit models of heart failure, includ-ing coronary artery occlusion models [114], have majoradvantages over other species. For example, the compositionof sarcomeric proteins in rabbits is similar to that inhumans, and the sarcolemmic reticulum contributes about70% of calcium elimination. In addition, the WHHLMIrabbit strain provides a model of spontaneous myocardialinfarction requiring no surgical intervention. This model wasdeveloped by selective breeding of coronary atherosclerosis-prone WHHL rabbits [124]. The main limitation of theWHHLMI model is that it does not feature plaque rupture,whereas in humans coronary plaque rupture and subsequentintravascular thrombosis are the major causes of acutemyocardial infarction. Despite this limitation, the model isvalid for the study of atherosclerosis-related heart complica-tions [113,125].An additional model of heart failure in large and smallanimals is pressure overload of the left ventricle, inducedby transverse aortic constriction in mice [110]andaorticbanding in rats and rabbits. Left ventricle hypertrophy canalso be recreated by ventricular pacing in dogs [115,118,126], valvular stenosis in rabbits [116], and renal arteryconstriction or aortic stenosis in rats, hamsters, mice, rabbitsand dogs [111,119].Another model of heart failure is the dilated cardiomy-opathy. Human dilated cardiomyopathy has been modeledin rabbits and pigs by inducing chronic tachycardia with apacemaker [121,127]. Transgenic mouse models, involvingmutations that predispose to dilated cardiomyopathy, havealso proved very useful. These models have identified anassociation of cytoskeletal and contractile proteins with thispathology, and very recently a transgenic model expressinga mutated cardiac alpha-actin gene was provided, in whichcalcium sensitivity of myofilaments is decreased and theexpression of calcium/calmodulin-dependent kinase IIdelta(CaMKIIdelta) is increased [112]. Inhibition of CaMKII-delta in these animals prevented the increase in p53 andapoptotic cardiomyocytes and ameliorated cardiac function.5. ConclusionAnimal models of cardiovascular disease yield importantinsights into the genetic basis of human cardiovasculardiseases and provide a test bed for pharmacological andtreatments. Nonetheless, investigators need to carefullyconsider their choice of model: no single method per-fectly recreates the human disease, and there are relatedconsiderations of cost, infrastructure and the requirementfor specialized personnel. Taking these considerations intoaccount, experimenters therefore need to select models thatbest reproduce the aspect of disease being investigated. Inparticular, when moving from bench to bedside it is essentialto test procedures in relevant models that yield highlyreproducible results, but despite these limitations, given therange of animal models available today it will always bepossible to devise an appropriate strategy, and animal modelsremain the best tools for advancing the understanding of themechanism of human cardiovascular disease.AcknowledgmentsAuthors’ work has been supported by Ministerio deCiencia y Tecnolog´ıa (SAF2007/63648, SAF2008/04629,SAF2009/11749, PI10/00072), CAM (S2006/GEN-0247), FIS(RECAVA RD06/0014/0035, PS09/00447), European Net-work (HEALTH F2-2008-200647), Euro Salud EUS2005-03565 and cvREMOD 091100. C. Zaragoza and C. Gomez-Guerrero contributed equally.References[1] G. S. Getz and C. A. 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AT A GLANCE Generating mouse models for biomedical research: technological advances Channabasavaiah B. Gurumurthy1,2 and Kevin C. Kent Lloyd3,4,* ABSTRACT Over the past decade, new methods and procedures have been developed to generate genetically engineered mouse models of human disease. This At a Glance article highlights several recent technical advances in mouse genome manipulation that have transformed our ability to manipulate and study gene expression in the mouse. We discuss how conventional gene targeting by homologous recombination in embryonic stem cells has given way to more refined methods that enable allele-specific manipulation in zygotes. We also highlight advances in the use of programmable endonucleases that have greatly increased the feasibility and ease of editing the mouse genome. Together, these and other technologies provide researchers with the molecular tools to functionally annotate the mouse genome with greater fidelity and specificity, as well as to generate new mouse models using faster, simpler and less costly techniques. KEY WORDS: CRISPR, Genome editing, Mouse, Mutagenesis Introduction Researchers are entering a new era of human disease modeling in animals. For many years now, the laboratory mouse (Mus musculus) has remained the quintessential research animal of choice for studying human biology, pathology and disease processes (Rosenthal and Brown, 2007; Lloyd et al., 2016). The mouse possesses numerous biological characteristics that make it the most commonly used animal in biomedical research for modeling human disease mechanisms; these characteristics include its short life 1 Developmental Neuroscience, Munroe Meyer Institute for Genetics and Rehabilitation, University of Nebraska Medical Center, Omaha, NE 68106-5915, USA. 2 Mouse Genome Engineering Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical Center, Omaha, NE 68106-5915, USA. 3Department of Surgery, School of Medicine, University of California, Davis, CA 95618, USA. 4Mouse Biology Program, University of California, Davis, CA 95618, USA. *Author for correspondence (kclloyd@ucdavis.edu) C.B.G., 0000-0002-8022-4033; K.C.K.L., 0000-0002-5318-4144 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. 1 © 2019. Published by The Company of Biologists Ltd | Disease Models & Mechanisms (2019) 12, dmm029462. doi:10.1242/dmm.029462 Disease Models & Mechanisms cycle, gestation period and lifespan, as well as its high fecundity and breeding efficiency (Silver, 2001). Another key advantage is its high degree of conservation with humans, as reflected in its anatomy, physiology and genetics (Justice and Dhillon, 2016). The highly conserved genetic homology that exists between mice and humans has justified the development of technologies to manipulate the mouse genome to create mouse models to reveal the genetic components of disease. It is important to note that, as technologies for genetic engineering and phenotypic analysis have advanced, some studies using mouse models have struggled to accurately predict human disease pathogenesis and clinical response to drug therapy (Perrin, 2014). For these reasons, it is essential to apply scientific principles of rigor and reproducibility (Kilkenny et al., 2010; Karp et al., 2015) when designing and conducting experiments to associate mouse genes with human phenotypes at a systems level (Perlman, 2016). Early mouse genetics research relied on mice having visible physical defects and readily measurable phenotypes, such as those caused by random spontaneous or induced mutations (Russell et al., 1979; Justice, 1999). This ‘forward genetics’ approach depends on the presence of a phenotype to guide the search for the underlying genetic mutation. With the advent of techniques that enabled molecular cloning and the use of recombinant DNA to efficiently manipulate mouse genomes, researchers no longer needed to search for a relevant phenotype. Instead, they could engineer a predetermined specific mutation into the mouse genome in real time in pluripotent mouse embryonic stem (ES) cells (Gordon and Ruddle, 1981; Gordon et al., 1980; Palmiter et al., 1982; Thomas and Capecchi, 1986, 1987). This ‘reverse genetics’ approach enabled scientists to study the phenotypic consequences of a known specific genetic mutation. This approach can generate ‘knockout’ mice (see Box 1 for a glossary of terms) by genetically manipulating the genome of ES cells, and then injecting the targeted cells into morulae or blastocysts (Box 1), which are then implanted into pseudopregnant female mice (Box 1). The resulting chimeric embryos develop into offspring that bear the desired gene deletion. After backcrossing to test for germline transmission of the knockout allele and subsequent intercrossing to achieve homozygosity, the phenotypic consequences of the mutation can be assessed. Phenotypes can also be assessed in transgenic mice (Box 1), which are generated by introducing an exogenous gene via microinjection into the one-cell-stage zygote. When successful, these genetic manipulations can also undergo germline transmission to the next generation (Palmiter et al., 1982; Brinster et al., 1989). With the sequencing of the mouse and human genomes (Venter et al., 2001; Mouse Genome Sequencing Consortium, 2002), attention soon turned to determining the function of protein-coding genes (Nadeau et al., 2001). A growing number (∼6000) of inherited disease syndromes (https://www.omim.org/statistics/geneMap) further motivated efforts to functionally annotate every human gene and to determine the genetic basis of rare, simple and common complex human diseases using mouse models. Mouse models are thus vitally important for elucidating gene function. Those that express the pathophysiology of human disease are an essential resource for understanding disease mechanisms, improving diagnostic strategies and for testing therapeutic interventions (Rosenthal and Brown, 2007; Bradley et al., 2012; Justice and Dhillon, 2016; Meehan et al., 2017). Even mouse models that only partially recapitulate the human phenotype, such as mutations in individual paralogs, can still provide important insights into disease mechanisms. In this At a Glance article, we review recent technological advances for generating new and improved mouse models for biomedical research. This article aims to update a previous poster published in this journal several years ago (Justice et al., 2011). This earlier article discussed the role of natural variation, random transgenesis, reverse genetics via ES-cell-derived knockouts, forward genetics via ethylnitrosurea (ENU)-induced chemical mutagenesis, and genetic manipulation using transposons in the generation of mouse models. Many technological advances have since emerged, leading to refinements and improvements in the generation of more precise mouse models. These new technologies overcome some of the limitations of earlier mouse models by adding specificity, reproducibility and efficiency to the generation of alleles that can expand our knowledge of disease pathogenesis. For example, the ability to generate mouse models that recapitulate the single-nucleotide variants (SNVs) found in humans will enable us to differentiate between disease-causing and disease-associated mechanisms (Hara and Takada, 2018). In the poster accompanying this article, we feature four areas of advancement: (1) conditional mutagenesis strategies in mouse ES cells; (2) gene function knockdown using RNA interference (RNAi); (3) targeted transgenesis in zygotes (Piedrahita et al., 1999; Shen et al., 2007) via homologous recombination (Box 1) in ES cells; and (4) the use of programmable endonucleases (Box 1) in zygotes, to edit and manipulate the mouse genome in ways not previously possible. These technologies represent a new paradigm in our ability to manipulate the mouse genome. However, as we discuss, these approaches are not without limitations. For example, the success of conditional mutagenesis can be hampered by poor gene-targeting efficiency in ES cells and by the limited production of germlinecompetent chimeras (Box 1) that can transmit the mutant allele to subsequent generations in their germline. Furthermore, protein expression can be highly variable following mRNA knockdown by RNAi, which can make experimental reproducibility a challenge. The major limitations of programmable endonucleases, the latest genome-editing tools, is mosaicism and their potential, albeit addressable, problem of inducing off-target mutations. Nonetheless, such pitfalls do not detract from the versatility that these newer technologies afford for manipulating the mouse genome. Conditional mutagenesis strategies in mouse ES cells The most common form of mouse genetic manipulation is the creation of gene knockout models. Gene-targeting in mouse ES cells was pioneered in the late 1980s and was first used to generate ubiquitous knockout models, in which the gene is deleted in every cell (Thomas and Capecchi, 1987; Thompson et al., 1989). We refer readers to the previous At a Glance article on modeling human disease in mice (Justice et al., 2011) for details on how to use gene targeting (Box 1) to generate simple deletion and/or conditional alleles (Box 1) in ES cells to generate whole-body and tissuespecific knockout mice, respectively. In this article, we focus on the generation of more-complex alleles in ES cells (Poster panel 1) that retain wild-type expression and are amenable to conditional, tissuespecific and/or time-dependent deletion. This approach is particularly necessary for manipulating the approximately 30% of genes that affect the viability of homozygous mutants when deleted (Dickinson et al., 2016). For example, embryonic lethality caused by the deletion of the coding regions of Mixl1 (Pulina et al., 2014), Erbb4 (Gassmann et al., 1995) or Brca1 (Xu et al., 1999) can be rescued by conditional mutagenesis. This generates models that can be used to investigate specific gene-dependent processes during mammalian embryogenesis (Pulina et al., 2014), neurodevelopment 2 AT A GLANCE Disease Models & Mechanisms (2019) 12, dmm029462. doi:10.1242/dmm.029462 Disease Models & Mechanisms (Golub et al., 2004) and breast cancer (Shakya et al., 2008) when combined with an appropriate Cre-expressing line that enables tissue- or developmental-stage-specific gene deletion (Dubois et al., 2006). The versatility of naturally occurring recombinase-enzyme– target-sequence systems, such as Cre/loxP (Box 1) and Flp/FRT, which derive from bacteria and yeast, respectively, have been adapted to create tools for manipulating mammalian genomes (Gu et al., 1994; Rajewsky et al., 1996; Dymecki, 1996). These tools have dramatically expanded the types and varieties of alleles that can be designed to study gene function in vivo (Dymecki, 1996; Nagy, 2000; Nern et al., 2011). A fundamental principle of conditional mutagenesis is the ability to efficiently and reliably convert a functional allele into a mutant one in a specific cell type (called tissue-specific conditional mutagenesis) and/or at a specific time point during development (called time-specific or ‘inducible’ conditional mutagenesis). Numerous strategies using recombinase-enzyme–target-sequence systems have been developed for conditional mutagenesis (Marth, 1996). Common to all these strategies is the use of short palindromic recombinase target sequences to flank a specific region of a gene (e.g. a critical coding exon common to all transcripts). Such sequences include the Cre-associated loxP sequence (to generate a ‘floxed’ allele) or the Flp-associated FRT sequence (to generate an ‘FRT’-flanked allele) (Bouabe and Okkenhaug, 2013). In the absence of the associated recombinase enzyme, these flanking sequences have no effect on normal transcription nor on the expression of the endogenous gene. However, when exposed to the recombinase, the flanking recombinase target sequences recombine with each other to excise or invert the critical coding exon, depending on their orientation and positioning (McLellan et al., 2017) (Poster panel 2A). In its simplest use, if two flanking recombinase target sequences are placed in an asymmetrical head-totail orientation, they will recombine to delete the intervening genetic sequence upon exposure to recombinase. Alternatively, if pairs of target sequences are positioned symmetrically in a head-to-head orientation, their recombination will invert the intervening sequence. If target sequences are located on different chromosomes, recombination results in a chromosomal translocation. There are different ways to elicit recombination. For example, as shown in Poster panel 2B, when a mouse that expresses a floxed allele is mated with a transgenic mouse that expresses the recombinase gene, its progeny will express the recombined allele (Gu et al., 1994). The tissue(s) in which the allele is recombined will depend on the expression pattern of the recombinase, i.e. where the promoter is activated to drive tissue-specific expression of the recombinase. Recombination can also be induced by the in vitro treatment of embryos or tissues with cell-permeable recombinase protein, or via the delivery of viral vectors that express the recombinase (Chambers et al., 2007; Lewandoski et al., 1997; Su et al., 2002). Recombinase activity can also be targeted to particular tissues by driving the expression of a recombinase from a cellspecific promoter. Recombinase expression can also be induced by expressing the recombinase from an inducible (e.g. drug-responsive) promoter (Sauer, 1998). The simplest example of the recombinase-enzyme–targetsequence system is shown in Poster panel 2C. This panel shows a molecular targeting construct in which the critical coding exon is flanked by loxP sites. The construct also contains a contiguous endogenous coding sequence of between 3 and 8 kb that is homologous to the wild-type allele. This construct is then introduced into ES cells, for example by electroporation, where it Box 1. Glossary Blastocyst: an early-stage (3.5 days post-fertilization) multicellular mouse embryo, which contains an inner mass of cells, a fluid-filled central cavity and an outer trophoblast cell layer. Chimera: a founder mouse that contains a mix of gene-targeted, embryonic stem (ES)-cell-derived cells and host blastocyst-derived cells, typically identified by the contribution of the two different genetic backgrounds of somatic cells to its coat color. Conditional alleles: an engineered allele that can be turned off (or on) in an exogenously controlled manner; for example, by recombinasemediated deletion of genomic sequences. Cre/loxP: a molecular recombination system that consists of a bacteriophage-derived recombinase protein (Cre) that binds to specific, non-mammalian, 34-nucleotide target sequences (loxP). Footprint-free point mutations: an induced mutation that is created without changes being made to untargeted sequences and without leaving exogenous DNA in place. Gene targeting: the methods used to make sequence changes to a specific gene rather than making random sequence changes; for example, gene targeting can be used to inactivate a gene. Homologous recombination: a natural DNA recombination process that occurs, for example, during meiosis and DNA repair, in which similar or identical DNA sequences are exchanged between two adjacent strands of DNA. Homology-directed repair (HDR): a DNA repair process involving the use of a single-stranded donor DNA template with short regions of homology (typically 30-60 bases long) as a donor template to fuse the cut ends of double-stranded DNA breaks created by programmable nucleases. Knock-down mouse: a genetically altered mouse in which gene expression is lowered or silenced by using RNAi to degrade the mRNA of that gene. Knock-in mouse: a genetically altered mouse in which a new mutation is introduced into an endogenous gene or an exogenous gene is introduced using genetic-engineering technologies. Knockout mouse: a genetically altered mouse in which an endogenous gene is deleted and/or inactivated using genetic-engineering technologies. loxP-stop-loxP: a commonly used DNA cassette, containing a stop codon flanked by loxP sites, included between the promoter and the coding sequences, to prevent expression of the coding sequence until the stop codon is excised by Cre-mediated recombination. Morula: an early-stage (2.5 days post-fertilization) pre-implantation mouse embryo, typically consisting of 4-8 blastomeres. Non-homologous end joining (NHEJ): a DNA repair mechanism that joins two DNA ends following a double-stranded break. Because the two ends are generally not homologous to each other, the process is named non-homologous end joining. Programmable endonuclease: an enzyme that, when coupled with molecular targeting elements (e.g. a guide RNA), creates site-specific double-stranded DNA breaks. Pronuclei: the structure in a one-cell-stage mouse embryo that contains the nucleus of the sperm and egg before these nuclei fuse. Pseudopregnant female: the state of ‘false’ pregnancy, created when a female in estrus is mated with a vasectomized male to induce the hormonal changes that simulate pregnancy in the absence of fertilized embryos. Recombinase-mediated cassette exchange (RMCE): a DNA integration strategy that uses site-specific recombinases, such as Cre or Flp, to exchange a DNA segment from one DNA molecule to another. Both the donor and target sequence are flanked by site-specific recombination sites, such as loxP or FRT. Double reciprocal recombination between these sites brings about DNA exchange. Safe-harbor sites: a genomic locus that, when genetically manipulated, neither interferes with the expression of an integrated transgene nor disrupts endogenous gene activity. Short hairpin (sh)RNA: a short or small RNA molecule with a hairpin loop used to silence gene expression by causing the degradation of the target mRNA. Small interfering (si)RNA: a short or small linear RNA molecule used to interfere with, or to silence, gene expression by causing the degradation of the target mRNA. Transgenic mouse: a genetically engineered mouse created by the pronuclear injection of recombinant DNA (transgene), which typically inserts at a random location in the genome. 3 AT A GLANCE Disease Models & Mechanisms (2019) 12, dmm029462. doi:10.1242/dmm.029462 Disease Models & Mechanisms then replaces, via homologous recombination, the endogenous wild-type allele (Hadjantonakis et al., 2008). The conditional allele can then undergo recombination upon exposure to the recombinase to delete the intervening critical coding exon, thereby inhibiting gene expression (null allele). Another strategy, termed ‘knockout-first’, uses a variation of gene targeting to create a highly versatile allele that combines both gene trap (Friedel and Soriano, 2010) and conditional gene targeting (Jovicićet al., 1990) to generate a lacZ-tagged knockout allele (Testa et al., 2004) (Poster panel 2D). The ‘knockout-first’ allele is generated by inserting an FRT-flanked gene-trap vector, which contains a splice-acceptor sequence upstream of a lacZ reporter gene and a strong polyadenylation stop sequence, into an upstream intron. This creates an in-frame fusion transcript that will disrupt the expression of the targeted allele. Additionally, an adjacent exon coding sequence is flanked with loxP sites (Rosen et al., 2015). This allele can then be converted into a null allele by Cre to abrogate gene expression or into a conditional allele by Flp, which can subsequently be converted by Cre into a null allele (Testa et al., 2004; Skarnes et al., 2011). The knockout-first strategy is versatile because it uses a single targeting vector to monitor gene expression using lacZ and tissue-specific gene function using Cre, thereby avoiding embryonic lethality. This strategy has been used effectively to enable the rapid and high-throughput production of thousands of gene knockouts in mouse ES cells in large-scale, genome-wide targeted mutagenesis programs, such as the International Knockout Mouse Consortium (IKMC) (Bradley et al., 2012). Hundreds of mutant mouse models of human genetic diseases have been generated using the knockout-first strategy, including models of skin abnormalities (Liakath-Ali et al., 2014), bone and cartilage disease (Freudenthal et al., 2016), and age-related hearing loss (Kane et al., 2012). Lastly, an elegant technique termed ‘conditionals by inversion’ (COIN) employs an inverted COIN module that contains a reporter gene (e.g. lacZ) flanked by mutant recombinase target sites (lox66 and lox71) positioned in a head-to-head orientation to enable inversion by Cre recombinase (Albert et al., 1995) inserted into the anti-sense strand of a target gene (Economides et al., 2013) (Poster panel 2E). Cre ‘flips’ the COIN module into the sense strand, interfering with and inhibiting target-gene transcription while activating the reporter. The COIN approach is particularly applicable to single-exon genes and to genes in which the exon– intron structure is not clearly defined. This approach has been used to model an angiogenesis defect in delta-like 4 (Dll4) knockout mice (Billiard et al., 2012) and to generate immunological phenotypes in interleukin 2 receptor, gamma chain (Il2rg) knockout mice (Economides et al., 2013). Gene expression knockdown using RNAi About two decades ago, researchers observed that the introduction of double-stranded RNA (dsRNA) that was homologous to a specific gene resulted in its posttranscriptional silencing (Fire et al., 1998). This dsRNA-induced gene silencing was termed RNA interference (RNAi), and it occurs via two main steps (Poster panel 3A). First, Dicer, an enzyme of the RNase III family of nucleases, processes the dsRNA into small double-stranded fragments termed siRNAs (small interfering RNAs; Box 1). Then, the siRNAs are incorporated into a nuclease complex called RISC (for RNAinduced silencing complex), which unwinds the siRNA and finds homologous target mRNAs using the siRNA sequence as a guide; this complex then cleaves the target mRNAs. In the early 2000s, some groups explored whether RNAi could be used to reduce (or ‘knock down’) gene expression in mice by creating transgenic mice that express siRNA (Poster panel 3B). The first proof-of-principle for gene knockdown was demonstrated by delivering lentivirus particles expressing siRNA into green fluorescent protein (GFP) transgenic mice to knock down GFP (Tiscornia et al., 2003). Subsequently, knockdown mice were generated using standard pronuclear injection of constructs that express short-hairpin RNAs (shRNA; Box 1) (Chang et al., 2004; Peng et al., 2006; Seibler et al., 2007; Dickins et al., 2007). Some examples of transgenic knockdown disease models include: an Abca1-deficient mouse line that mimics Tangier disease (Chang et al., 2004); insulin receptor (Insr)-knockdown mice that develop severe hyperglycemia within 7 days (Seibler et al., 2007); and the reversible knockdown of Trp53 as a model useful for tumor regression studies (Dickins et al., 2007). The advantage of the RNAi knockdown strategy over traditional methods for generating knockout mice is that it provides a rapid and inexpensive approach by which to selectively and, in some cases, reversibly block the translation of a transcript. Although knockdown models can be generated more quickly and cheaply than genetargeted knockout models (Liu, 2013), a key disadvantage of a knockdown is that transcript inhibition can be variable and transient, and therefore less reliable and reproducible than a knockout. The effects of random insertion, together with varying levels of RNAi in different cells within a tissue, were among the most common pitfalls associated with using RNAi technology to modify mouse gene expression (Peng et al., 2006; Yamamoto-Hino and Goto, 2013). Because of such challenges, and due to the lack of success in generating reliable transgenic RNAi models, this approach did not gain the expected popularity. Alternative strategies were developed to overcome the effect of randomly inserted RNAi constructs by targeting the knockdown cassettes to safe-harbor sites (Box 1), such as the Gt(ROSA)26Sor locus (Kleinhammer et al., 2010) or the Cola1 locus (Premsrirut et al., 2011). These strategies also include making the system modular by incorporating features such as: (i) the Flp-FRT recombinase-mediated cassette exchange (RMCE; Box 1), which facilitates the insertion of a single-copy expression cassette; (ii) a fluorescence reporter that enables gene expression analysis; (iii) microRNA (miRNA) architectures, such as miR30 with reduced general toxicity (McBride et al., 2008); and (iv) tetracycline-inducible elements to enable the expression of the RNAi cassettes upon doxycycline administration (Chang et al., 2004; Seibler et al., 2007). A few models that are useful for cancer research have been generated using these approaches, such as Pax5 and eIF4F knockdown models for leukemia (Lin et al., 2012; Liu et al., 2014). However, interest in generating knockdown models, as well as in using ES-cell-based gene targeting, began to wane with the development of programmable nuclease technologies (as discussed later). More recently, an elegant approach that combines the use of the RNA-guided Cas9 nuclease system with RNAi technology has been developed to generate knockdown mouse models by inserting the knockdown cassettes into the intronic sites of endogenous genes (Miura et al., 2015). With this method, a single-copy artificial miRNA against the Otx2 gene was inserted into intron 6 of the Eef2 gene to knock down Otx2 in mid-gestation mouse embryos. This strategy was also used to conditionally activate knockdown cassettes using unidirectional recombinase-mediated inversion of the shRNA cassette. The Miura et al. method offers a feasible and simple strategy to generate gene knockdown models because: (i) it uses an endogenous promoter, unlike other knockdown approaches that require an exogenous promoter to drive the RNAi cassette; (ii) the knockdown cassette is inserted as a single copy at a known 4 AT A GLANCE Disease Models & Mechanisms (2019) 12, dmm029462. doi:10.1242/dmm.029462 Disease Models & Mechanisms site in the genome, unlike approaches that randomly insert the cassette with no control over the number of copies inserted or the number of genomic insertion sites; and (iii) the transgene is not susceptible to silencing, in contrast to other transgenes that are often silenced following random genomic integration. Pronuclear injection-based transgenesis Traditional transgenic methods developed over three decades ago involve the injection of linearized DNA expression cassettes into fertilized zygotes (Gordon et al., 1980; Palmiter et al., 1982) (Poster panel 4A). Some of the most commonly used transgenic DNA expression cassettes include: (i) cDNA encoding the wild-type or mutant allele; (ii) inducible reporter cassettes, such as the loxP-stoploxP reporter (Box 1), that incorporate markers such as lacZ or the fluorescent reporters GFP, red fluorescent protein (RFP) or tdTomato; (iii) recombinases, such as Cre (Gu et al., 1994), tamoxifen-inducible Cre (CreERT2) (Feil et al., 1996) and Flp (Dymecki, 1996); and (iv) transcriptional inducers, such as tetracycline transactivators (tTA) or reverse tetracycline transactivators (rtTA) (Gossen and Bujard, 1992). To produce transgenic mice, a DNA construct is microinjected into the pronuclei (Box 1) of one-cell-stage zygotes (Bockamp et al., 2008). All or part of the injected DNA then inserts randomly at one or more genomic loci as either a single or as multiple (e.g. tandem-repeat) copies. The suitability of this approach for generating animal models is limited by the uncertainty of obtaining a desired level of gene expression due to the random nature of transgene insertion and copy number (Chiang et al., 2012). As a result, ES-cell-based methods were developed to target expression cassettes (such as those encoding Cre) into a specific locus in the genome; for example, the Gt(ROSA)26Sor locus, which enables the ubiquitous expression of an inserted transgene (Soriano, 1999). Depending on the construct and insertion site, transgene expression could be driven by a target gene’s endogenous promoter and/or by other regulatory elements (Rickert et al., 1997). In this way, an intact, single-copy transgene becomes integrated into a predetermined genomic location in ES cells via homologous recombination, thereby optimizing transgene expression (Rickert et al., 1997; Soriano, 1999). The targeted ES cells are then introduced into morulae or blastocysts, as previously explained, before being implanted into pseudopregnant females. Although this approach overcomes some of the constraints inherent to random transgenesis (such as high variability of gene expression, and difficulty in obtaining the desired transgene expression patterns and levels), homologous recombination has technical hurdles of its own that make it expensive, labor intensive and time consuming. In addition, germline transmission of the exogenous allele can fail, creating a frustrating struggle for researchers who need to reliably and regularly manipulate the mouse genome (Ohtsuka et al., 2012a). Another disadvantage of the ES cell targeting approach is that ES cell genomes do not always remain stable in culture, and can undergo changes before and after gene targeting (Liang et al., 2008). The recently developed targeted transgenic technologies enable the integration of single-copy transgenes at specific loci in the genome, directly via pronuclear injection. In pioneering work, Masato Ohtsuka and co-workers developed a method called pronuclear injection-based targeted transgenesis (PITT) (Ohtsuka et al., 2010), which allows a single copy of a complete transgene to be precisely inserted at a desired genomic locus in the zygote (Poster panel 4B). The PITT method involves two steps. First, a landing pad (for example, a cassette containing a combination of mutant loxP sites) is inserted at a defined locus in ES cells to generate a ‘seed’ mouse strain. Second, the PITT components – a donor plasmid containing the DNA of interest (DOI) and a Cre source (either plasmid or mRNA) – are injected into fertilized eggs collected from the seed strain mice. The DOI inserts at the landing pad via recombination-mediated cassette exchange (RMCE). The landing pad and the donor DNA contain compatible sequence elements that enable the donor DNA to insert precisely into the target locus. In the first report (Ohtsuka et al., 2010), the authors employed a wellestablished Cre-loxP system (as the components of the landing pad and the donor plasmid elements) to achieve RMCE. Soon after the first description of the PITT technology, another group reported a similar approach using the PhiC31 integrase and attP/B system, which correspond to the landing pad components and donor plasmid elements (Tasic et al., 2011). This modified method to achieve targeted transgenesis was named Targatt™ (Chen-Tsai et al., 2014). The main advantages of the various targeted transgenesis methods that use either Cre-loxP recombination or PhiC31-attP/B integration, are that: (i) they overcome the problems associated with random transgene insertion, such as fragmented insertion of the transgenes, multicopy insertions, transgene silencing or interference in the expression of the endogenously disrupted gene; and (ii) they resolve the time and cost limitations associated with ES-cell-based approaches by targeting DNA cassettes to specific sites in the genome. In initial reports of the PITT method, the Cre recombinase was encoded by a plasmid, and the plasmid DNA was injected into the pronuclei of zygotes together with the donor DNA. This method has since been improved by: (i) the use of Cre mRNA instead of plasmid DNA, which was done because plasmid DNA needs to be transcribed, which delays the expression of Cre, by which time the donor DNA might have degraded (Ohtsuka et al., 2012b); (ii) the development of new PITT-compatible donor vectors (Ohtsuka et al., 2012b); and (iii) the development of a seed mouse strain that contains both Cre-loxP and PhiC31-attP/B cassette insertion systems, providing researchers with the flexibility to use either (Ohtsuka et al., 2015). In this format, multiple different PITT donor plasmids can be included in the microinjection mix: any one of these donors can be inserted at the landing pad in separate founder mice, resulting in independent transgenic mouse lines generated in a single session of microinjection. These latest technical tools, dubbed ‘improved PITT’ (i-PITT), allow up to three transgenic mouse lines to be generated simultaneously, such that each line has a different DOI after a single microinjection session (Ohtsuka et al., 2015). The PITT technology is reviewed in detail in Ohtsuka et al., 2012a and a comprehensive list of available PITT tools was recently described (Schilit et al., 2016). The PITT/i-PITT approaches have been used to generate many reliable single-copy transgenic reporter mouse lines that are useful for disease research, including in neuroscience (Madisen et al., 2015) and nephrology (Tsuchida et al., 2016). For example, Tsuchida et al. (2016) reported generating a nephrin-promoter-driven EGFP transgenic mouse model; they further showed that cultured glomeruli from this model serve as tools to screen for compounds that enhance nephrinpromoter activity. Although PITT strategies have overcome the limitations of random transgenesis, a major pitfall of this approach is that custom PITT seed mouse strains need to be generated for a given locus and maintained as breeder colonies as zygote donors for targeted transgenesis. Despite the technical advances in genetic engineering over the past four decades, one recent and remarkable technical breakthrough is rapidly superseding nearly all of these advances: programmable endonucleases. 5 AT A GLANCE Disease Models & Mechanisms (2019) 12, dmm029462. doi:10.1242/dmm.029462 Disease Models & Mechanisms Programmable endonucleases for genome editing Programmable endonucleases bypass the classical ES-cell-based gene-targeting steps to engineer a precise and heritable mutation at a specific site in the genome. Injection directly into one- or two-cellstage embryos enables the germline modification of a specific genetic locus without the need for the three complex steps above. Programmable endonucleases can introduce genetic mutations in one of two ways (Joung and Sander, 2012; Gaj et al., 2013; Sander and Joung, 2014; Cox et al., 2015). They can cause: (i) imprecise, error-prone DNA repair as a result of non-homologous end joining (NHEJ; Box 1) of the cleaved DNA ends; or (ii) the precise repair of cleaved DNA ends by homology-directed repair (HDR; Box 1) via the co-injection of a DNA repair template. Nonetheless, the imprecise insertion of the donor DNA can still occur in HDRmediated repair. The development of programmable endonucleases for genome editing has opened up a whole new set of technical possibilities to create animal models for biomedical research using virtually any suitable species. There are four major platforms that employ programmable endonucleases, which were initially discovered in microbiology research applications (Chevalier and Stoddard, 2001; Li et al., 1992; Mojica and Garrett, 2013; Mojica et al., 1993; Römer et al., 2007) and have since been repurposed for editing the genomes of higher animals, including mice. They are, in the order they were developed: homing endonucleases (HEs); zinc-finger nucleases (ZFNs); transcription activator-like effector nucleases (TALENs); and the clustered regularly interspaced short palindromic repeats/CRISPRassociated 9 (CRISPR/Cas9) system (Poster panel 5). Common to all four programmable endonuclease platforms is their sequencespecific nuclease activity, which allows researchers to cleave DNA at a specific target site for genome editing (Joung and Sander, 2012; Gaj et al., 2013; Sander and Joung, 2014; Cox et al., 2015). The HEs were among the first of the endonucleases (Rouet et al., 1994) to be used for genome manipulation. Although HEs were shown to increase gene-targeting efficiency in ES cells (Smih et al., 1995), there is little evidence to suggest that they have been used successfully to genetically engineer mutant mice. This is probably because of the numerous steps required to design and construct HEs to target specific genomic sites, and because only a small number of genomic sites could be targeted. The ZFNs, unlike HEs, offered greater flexibility as they are easier to engineer and can target more genomic locations than can HEs (Poster panel 5). From 2002 onwards, ZFNs became more widely used than HEs, especially as a research tool in various organisms, including flies, fish and plants (Urnov et al., 2010; Carroll, 2011). The first ZFN-modified mutant mouse models were described in 2010 by Carbery and co-workers via the direct injection of ZFNs that target and inactivate Mdr1a, Jag1 and Notch3 (Carbery et al., 2010). Nevertheless, the technical complexity of building ZFNs, and intellectual property restrictions, limited their widespread adaptability. TALENs, the next set of programmable nucleases, were developed in 2010 and overcame many of the limitations of HEs and ZFNs. TALENs were simpler, easier to build and could be used to target a greater number of genomic sites than could HEs or ZFNs, and thus were immediately adopted by hundreds of labs as research tools. The first mutant mouse models using TALENs were developed by Sung and co-workers in 2013 via the direct injection of TALENs that targeted Pibf1 and Sepw1 to inactivate them (Sung et al., 2013). At the time when ZFNs and TALENs were being developed, each platform proved to be quite versatile and superior to the previously available genetic engineering tools. Then came the development of the CRISPR/Cas9 genome editing tool in late 2012 and early 2013 (Jinek et al., 2012; Cong et al., 2013; Mali et al., 2013) (Poster panel 5). A series of papers from multiple groups, published within a few months of each other, demonstrated that dsDNA breaks at specific sites in the genome could be generated with very high efficiency in mammalian cells by using guide RNAs complementary to the target site and the Cas9 nuclease (Jinek et al., 2012, 2013; Mali et al., 2013; Cong et al., 2013; Cho et al., 2013). Within just a few months, some groups demonstrated that the RNA-guided Cas9 nuclease system could be used to rapidly generate mutant mouse models (Shen et al., 2013; Wang et al., 2013). Since then, the RNA-guided Cas9 nuclease system has almost completely superseded all other technologies for genome editing. A direct comparison of the RNA-guided Cas9 nuclease system with the previous nuclease-based platforms (HEs, ZFNs and TALENs) clearly shows that it has several advantages (Sander and Joung, 2014; Porteus, 2015; Woolf et al., 2017). These include its simplicity of use, lower cost and higher efficiency. The RNAguided Cas9 nuclease system is constantly being improved to make it increasingly efficient and versatile, including optimizing and improving the efficiency of existing Cas nucleases (Kleinstiver et al., 2016; Slaymaker et al., 2016), and the development of novel Cas nucleases (Shmakov et al., 2015; Zetsche et al., 2015). The RNA-guided Cas9 nuclease system is considered a ‘disruptive’ technology because it is quickly making previously wellestablished and fully developed technologies outdated. In recent years, researchers have come to prefer this approach over ES-cellbased gene-targeting methods (Burgio, 2018; Skarnes, 2015) because RNA-guided Cas9 nuclease approaches are relatively quicker, less expensive and less cumbersome. The versatility of the RNA-guided Cas9 nuclease system allows researchers to engineer and edit the genome in ways that were previously not possible using non-nuclease-based approaches (Poster panel 5). This includes the ease and speed with which researchers can induce a footprint-free point mutation (Box 1) (Inui et al., 2014; Gurumurthy et al., 2016a). Many human disease conditions are caused by subtle genetic changes, such as point mutations, or by the addition or deletion of a few nucleotides (Gonzaga-Jauregui et al., 2012). Developing animal models of such subtle genetic changes, by using ES-cell-based targeting approaches, inevitably requires the addition of other genetic elements near the vicinity of the genetic change [such as a drug selection marker (neomycin or puromycin) and recombinase elements (such as loxP or FRT sites)]. By contrast, the RNA-guided Cas9 nuclease system can generate animal models with subtle genetic changes with high precision, rapidly, efficiently and without leaving any residual genetic alterations. Compared to previous methods, this capability represents a significant advance in murine genome editing for human disease modeling. The RNAguided Cas9 nuclease tool has also facilitated the generation of multiple mutant mouse models in a single experiment by inducing dsDNA breaks at multiple target sites, resulting in several different gene disruption models (Wang et al., 2013). The RNA-guided Cas9 nuclease system also enables the generation of mutant mouse models on genetic backgrounds that were not amenable to being genetically manipulated with earlier approaches, such as the immunodeficient NOD/Scid-ILgamma (NSG) strain (Li et al., 2014). The RNAguided Cas9 nuclease system has also become a powerful tool for both forward and reverse genetics (Gurumurthy et al., 2016c), generating models that are relevant for many diseases, including cancer (Platt et al., 2014). Several recent review articles discuss the Cas9-nuclease-generated mouse models for different disease types, including for cancer (Mou et al., 2015; Roper et al., 2017), cardiovascular diseases (Miano et al., 2016), neurodegenerative 6 AT A GLANCE Disease Models & Mechanisms (2019) 12, dmm029462. doi:10.1242/dmm.029462 Disease Models & Mechanisms diseases (Yang et al., 2016) and kidney diseases (Higashijima et al., 2017). In addition, several reviews on Cas9-nuclease-generated models have been recently published that discuss their human disease relevance (Dow, 2015; Tschaharganeh et al., 2016; Cai et al., 2016; Yang et al., 2016; Birling et al., 2017). Despite its advantages, the RNA-guided Cas9 nuclease system poses challenges, such as mosaicism (Yen et al., 2014) and offtarget effects. If one of the two haploid genomes in the one-cellstage zygote is not cleaved before the zygote divides, or if Cas9 activity persists at the two-cell or later stages, additional mutant alleles can be generated, resulting in more than three mutant alleles in the developing offspring. Consequently, as many as six or more types of alleles were detected in one founder (G0) mouse (Li et al., 2013). It is therefore essential to genotype F1 offspring to identify a desired mutant allele. This mosaicism can also be considered an advantage because multiple different alleles can be segregated and used as separate mutant models. For example, the same founder mouse could contain a complete insertion deletion (indel) allele and the foreign cassette knock-in allele; each can be used for different research applications. Because the Cas9 target sequence is only 23 nucleotides long, including the protospacer adjacent motif, it is likely that imperfect target-matching sequences are present elsewhere in the genome that contain one or a few mismatches. Cas9 can potentially bind to such imperfect target sites and thus generate dsDNA breaks and indels at those sites. Indel mutations in off-target sites can have confounding effects in mouse phenotyping experiments. However, off-target effects are not considered a major concern because they: (i) are generally negligible in mice (Iyer et al., 2015); and (ii) can be segregated during mouse breeding. Another recent study, now retracted, reported the presence of high rates of off-target effects in Cas9 engineered mice (Schaefer et al., 2017); however, this report’s experimental design and interpretations have been questioned by the scientific community (Kim et al., 2018; Lescarbeau et al., 2018; Nutter et al., 2018; Wilson et al., 2018). A current challenge to the broader use of RNA-guided Cas9 nuclease is the inability to use it to insert large fragments of DNA reliably and efficiently. Because most genetic-engineering approaches in mice involve the insertion of engineered DNA cassettes, efforts are underway to improve the ‘knock-in’ capabilities of this system. While a few RNA-guided Cas9 nuclease strategies have been modified to support the insertion of new cassettes (Aida et al., 2015; Maruyama et al., 2015; Sakuma et al., 2016), including a strategy that combines PITT and RNA-guided Cas9 nuclease approaches (Quadros et al., 2015), none has yet been successfully adapted for the routine engineering of the mouse genome. A report from Ohtsuka’s group, which used long single-stranded DNA (lssDNA) donors (generated via in vitro transcription and reverse transcription), demonstrated that lssDNAs could serve as efficient donors for insertion at the Cas9 cleavage sites (Miura et al., 2015). Another report, which used lssDNAs purified from nicked plasmids to create rat knock-in models, also demonstrated that the lssDNA donor strategy could be a reliable approach for creating insertion alleles (Yoshimi et al., 2016). More recent reports show that coinjecting lssDNA donors with commercially available CRISPR ribonucleoprotein complexes (instead of the previous formats of Cas9 mRNA and sgRNAs), offers a highly robust and efficient strategy for insertion alleles in a method termed Easi-CRISPR (efficient additions with ssDNA inserts-CRISPR) (Quadros et al., 2017; Miura et al., 2017). RNA-guided Cas9 nuclease reagents have also been delivered into zygotes via electroporation of RNA and/or of ribonucleoproteins (Chen et al., 2016; Hashimoto and Takemoto, 2015; Qin et al., 2015). The ability to deliver RNA-guided Cas9-nuclease gene-editing reagents into several zygotes at once overcomes the need to inject each individual zygote, one at a time, and greatly simplifies the process of generating mouse models. Furthermore, electroporation is less damaging to embryos than microinjection (Chen et al., 2016; Hashimoto and Takemoto, 2015; Qin et al., 2015). Another advance in delivering the RNA-guided Cas9 nuclease system is a method called GONAD (genome editing via oviductal nucleic acids delivery). This procedure delivers Cas9 reagents to embryos in the oviduct using electroporation (Takahashi et al., 2015; Gurumurthy et al., 2016b; Sato et al., 2016; Ohtsuka et al., 2018). Unlike standard approaches, this method does not require any of the three major steps of animal transgenesis: zygote isolation from a female donor; ex vivo handling of zygotes (involving either microinjection or electroporation); and the transfer of zygotes to a pseudopregnant female mouse. This approach requires surgical skills that are equivalent to performing the oviductal transfer of embryos. The GONAD method can be used to generate knockout mice (Takahashi et al., 2015), and, by using the so-called improved-GONAD (i-GONAD), more complex animal models, such as knock-ins and large-deletion models, can be generated at an efficiency similar to the microinjection-based methods (Ohtsuka et al., 2018). The i-GONAD method also uses only a third of the mice used in standard microinjection or in ex vivo zygote electroporation methods (Ohtsuka et al., 2018). These methods need not be limited to centralized facilities, sophisticated equipment or highly skilled technical personnel. It is thought that the technical advances such as Easi-CRISPR and i-GONAD have the potential to entirely reshape the traditional route of generating modified alleles in mice if the techniques are widely adopted by many research groups and by transgenic core facilities (Burgio, 2018). Concluding remarks and future perspectives Recent technological breakthroughs have enabled very rapid changes in the way we generate genetically altered mouse models. Most notably, the RNA-guided Cas9 nuclease system is assuming a key role in shaping this new technological landscape. While the use of the RNA-guided Cas9 nuclease system has transformed and eclipsed traditional transgenic technologies in many ways, challenges remain, including the inability to insert large DNA constructs to generate a knock-in mouse (Box 1) with reporter, conditional or humanized alleles, or to engineer chromosomal rearrangements and other complex alleles easily, routinely and efficiently. Genetic manipulation also underpins the ongoing efforts to elucidate the functional roles of every gene in the mouse genome, as a first step to understanding the role of ‘disease alleles’ identified by the exome and genome sequencing of human patients. Genomic and precision medicine depends on our ability to differentiate benign from pathogenic variant alleles, and disease-causing alleles from the longer list of disease-associated ones. Genetic manipulation of the mouse genome is thus essential for understanding gene function and for uncovering the genetic and molecular basis of human disease, leading to improved diagnostic accuracy, development of targeted therapeutics and the implementation of effective prevention strategies. At a glance A high-resolution version of the poster is available for downloading in the online version of this article at http://dmm.biologists.org/content/12/1/dmm029462/F1. poster.jpg. References Aida, T., Chiyo, K., Usami, T., Ishikubo, H., Imahashi, R., Wada, Y., Tanaka, K. F., Sakuma, T., Yamamoto, T. and Tanaka, K. (2015). Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice. 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AT A GLANCEGenerating mouse models for biomedical research: technological advancesChannabasavaiah B. Gurumurthy, Kevin C. Kent LloydDisease Models & Mechanisms 2019 12: dmm029462 doi: 10.1242/dmm.029462 Published 8 January 2019

ABSTRACT

Over the past decade, new methods and procedures have been developed to generate genetically engineered mouse models of human disease. This At a Glance article highlights several recent technical advances in mouse genome manipulation that have transformed our ability to manipulate and study gene expression in the mouse. We discuss how conventional gene targeting by homologous recombination in embryonic stem cells has given way to more refined methods that enable allele-specific manipulation in zygotes. We also highlight advances in the use of programmable endonucleases that have greatly increased the feasibility and ease of editing the mouse genome. Together, these and other technologies provide researchers with the molecular tools to functionally annotate the mouse genome with greater fidelity and specificity, as well as to generate new mouse models using faster, simpler and less costly techniques.

Introduction

Researchers are entering a new era of human disease modeling in animals. For many years now, the laboratory mouse (Mus musculus) has remained the quintessential research animal of choice for studying human biology, pathology and disease processes (Rosenthal and Brown, 2007Lloyd et al., 2016). The mouse possesses numerous biological characteristics that make it the most commonly used animal in biomedical research for modeling human disease mechanisms; these characteristics include its short life cycle, gestation period and lifespan, as well as its high fecundity and breeding efficiency (Silver, 2001). Another key advantage is its high degree of conservation with humans, as reflected in its anatomy, physiology and genetics (Justice and Dhillon, 2016).

The highly conserved genetic homology that exists between mice and humans has justified the development of technologies to manipulate the mouse genome to create mouse models to reveal the genetic components of disease. It is important to note that, as technologies for genetic engineering and phenotypic analysis have advanced, some studies using mouse models have struggled to accurately predict human disease pathogenesis and clinical response to drug therapy (Perrin, 2014). For these reasons, it is essential to apply scientific principles of rigor and reproducibility (Kilkenny et al., 2010Karp et al., 2015) when designing and conducting experiments to associate mouse genes with human phenotypes at a systems level (Perlman, 2016).

Early mouse genetics research relied on mice having visible physical defects and readily measurable phenotypes, such as those caused by random spontaneous or induced mutations (Russell et al., 1979Justice, 1999). This ‘forward genetics’ approach depends on the presence of a phenotype to guide the search for the underlying genetic mutation. With the advent of techniques that enabled molecular cloning and the use of recombinant DNA to efficiently manipulate mouse genomes, researchers no longer needed to search for a relevant phenotype. Instead, they could engineer a pre-determined specific mutation into the mouse genome in real time in pluripotent mouse embryonic stem (ES) cells (Gordon and Ruddle, 1981Gordon et al., 1980Palmiter et al., 1982Thomas and Capecchi, 19861987). This ‘reverse genetics’ approach enabled scientists to study the phenotypic consequences of a known specific genetic mutation. This approach can generate ‘knockout’ mice (see Box 1 for a glossary of terms) by genetically manipulating the genome of ES cells, and then injecting the targeted cells into morulae or blastocysts (Box 1), which are then implanted into pseudopregnant female mice (Box 1). The resulting chimeric embryos develop into offspring that bear the desired gene deletion. After backcrossing to test for germline transmission of the knockout allele and subsequent intercrossing to achieve homozygosity, the phenotypic consequences of the mutation can be assessed. Phenotypes can also be assessed in transgenic mice (Box 1), which are generated by introducing an exogenous gene via microinjection into the one-cell-stage zygote. When successful, these genetic manipulations can also undergo germline transmission to the next generation (Palmiter et al., 1982Brinster et al., 1989).

With the sequencing of the mouse and human genomes (Venter et al., 2001Mouse Genome Sequencing Consortium, 2002), attention soon turned to determining the function of protein-coding genes (Nadeau et al., 2001). A growing number (∼6000) of inherited disease syndromes (https://www.omim.org/statistics/geneMap) further motivated efforts to functionally annotate every human gene and to determine the genetic basis of rare, simple and common complex human diseases using mouse models. Mouse models are thus vitally important for elucidating gene function. Those that express the pathophysiology of human disease are an essential resource for understanding disease mechanisms, improving diagnostic strategies and for testing therapeutic interventions (Rosenthal and Brown, 2007Bradley et al., 2012Justice and Dhillon, 2016Meehan et al., 2017). Even mouse models that only partially recapitulate the human phenotype, such as mutations in individual paralogs, can still provide important insights into disease mechanisms.

In this At a Glance article, we review recent technological advances for generating new and improved mouse models for biomedical research. This article aims to update a previous poster published in this journal several years ago (Justice et al., 2011). This earlier article discussed the role of natural variation, random transgenesis, reverse genetics via ES-cell-derived knockouts, forward genetics via ethylnitrosurea (ENU)-induced chemical mutagenesis, and genetic manipulation using transposons in the generation of mouse models. Many technological advances have since emerged, leading to refinements and improvements in the generation of more precise mouse models. These new technologies overcome some of the limitations of earlier mouse models by adding specificity, reproducibility and efficiency to the generation of alleles that can expand our knowledge of disease pathogenesis. For example, the ability to generate mouse models that recapitulate the single-nucleotide variants (SNVs) found in humans will enable us to differentiate between disease-causing and disease-associated mechanisms (Hara and Takada, 2018).

In the poster accompanying this article, we feature four areas of advancement:

(1) conditional mutagenesis strategies in mouse ES cells;

(2) gene function knockdown using RNA interference (RNAi);

(3) targeted transgenesis in zygotes (Piedrahita et al., 1999Shen et al., 2007) via homologous recombination (Box 1) in ES cells; and

(4) the use of programmable endonucleases (Box 1) in zygotes, to edit and manipulate the mouse genome in ways not previously possible.

These technologies represent a new paradigm in our ability to manipulate the mouse genome. However, as we discuss, these approaches are not without limitations. For example, the success of conditional mutagenesis can be hampered by poor gene-targeting efficiency in ES cells and by the limited production of germline-competent chimeras (Box 1) that can transmit the mutant allele to subsequent generations in their germline. Furthermore, protein expression can be highly variable following mRNA knockdown by RNAi, which can make experimental reproducibility a challenge. The major limitations of programmable endonucleases, the latest genome-editing tools, is mosaicism and their potential, albeit addressable, problem of inducing off-target mutations. Nonetheless, such pitfalls do not detract from the versatility that these newer technologies afford for manipulating the mouse genome.

Box 1. Glossary

Blastocyst: an early-stage (3.5 days post-fertilization) multicellular mouse embryo, which contains an inner mass of cells, a fluid-filled central cavity and an outer trophoblast cell layer.

Chimera: a founder mouse that contains a mix of gene-targeted, embryonic stem (ES)-cell-derived cells and host blastocyst-derived cells, typically identified by the contribution of the two different genetic backgrounds of somatic cells to its coat color.

Conditional alleles: an engineered allele that can be turned off (or on) in an exogenously controlled manner; for example, by recombinase-mediated deletion of genomic sequences.

Cre/loxP: a molecular recombination system that consists of a bacteriophage-derived recombinase protein (Cre) that binds to specific, non-mammalian, 34-nucleotide target sequences (loxP).

Footprint-free point mutations: an induced mutation that is created without changes being made to untargeted sequences and without leaving exogenous DNA in place.

Gene targeting: the methods used to make sequence changes to a specific gene rather than making random sequence changes; for example, gene targeting can be used to inactivate a gene.

Homologous recombination: a natural DNA recombination process that occurs, for example, during meiosis and DNA repair, in which similar or identical DNA sequences are exchanged between two adjacent strands of DNA.

Homology-directed repair (HDR): a DNA repair process involving the use of a single-stranded donor DNA template with short regions of homology (typically 30-60 bases long) as a donor template to fuse the cut ends of double-stranded DNA breaks created by programmable nucleases.

Knock-down mouse: a genetically altered mouse in which gene expression is lowered or silenced by using RNAi to degrade the mRNA of that gene.

Knock-in mouse: a genetically altered mouse in which a new mutation is introduced into an endogenous gene or an exogenous gene is introduced using genetic-engineering technologies.

Knockout mouse: a genetically altered mouse in which an endogenous gene is deleted and/or inactivated using genetic-engineering technologies.

loxP-stop-loxP: a commonly used DNA cassette, containing a stop codon flanked by loxP sites, included between the promoter and the coding sequences, to prevent expression of the coding sequence until the stop codon is excised by Cre-mediated recombination.

Morula: an early-stage (2.5 days post-fertilization) pre-implantation mouse embryo, typically consisting of 4-8 blastomeres.

Non-homologous end joining (NHEJ): a DNA repair mechanism that joins two DNA ends following a double-stranded break. Because the two ends are generally not homologous to each other, the process is named non-homologous end joining.

Programmable endonuclease: an enzyme that, when coupled with molecular targeting elements (e.g. a guide RNA), creates site-specific double-stranded DNA breaks.

Pronuclei: the structure in a one-cell-stage mouse embryo that contains the nucleus of the sperm and egg before these nuclei fuse.

Pseudopregnant female: the state of ‘false’ pregnancy, created when a female in estrus is mated with a vasectomized male to induce the hormonal changes that simulate pregnancy in the absence of fertilized embryos.

Recombinase-mediated cassette exchange (RMCE): a DNA integration strategy that uses site-specific recombinases, such as Cre or Flp, to exchange a DNA segment from one DNA molecule to another. Both the donor and target sequence are flanked by site-specific recombination sites, such as loxP or FRT. Double reciprocal recombination between these sites brings about DNA exchange.

Safe-harbor sites: a genomic locus that, when genetically manipulated, neither interferes with the expression of an integrated transgene nor disrupts endogenous gene activity.

Short hairpin (sh)RNA: a short or small RNA molecule with a hairpin loop used to silence gene expression by causing the degradation of the target mRNA.

Small interfering (si)RNA: a short or small linear RNA molecule used to interfere with, or to silence, gene expression by causing the degradation of the target mRNA.

Transgenic mouse: a genetically engineered mouse created by the pronuclear injection of recombinant DNA (transgene), which typically inserts at a random location in the genome.

Conditional mutagenesis strategies in mouse ES cells

The most common form of mouse genetic manipulation is the creation of gene knockout models. Gene-targeting in mouse ES cells was pioneered in the late 1980s and was first used to generate ubiquitous knockout models, in which the gene is deleted in every cell (Thomas and Capecchi, 1987Thompson et al., 1989). We refer readers to the previous At a Glance article on modeling human disease in mice (Justice et al., 2011) for details on how to use gene targeting (Box 1) to generate simple deletion and/or conditional alleles (Box 1) in ES cells to generate whole-body and tissue-specific knockout mice, respectively. In this article, we focus on the generation of more-complex alleles in ES cells (Poster panel 1) that retain wild-type expression and are amenable to conditional, tissue-specific and/or time-dependent deletion. This approach is particularly necessary for manipulating the approximately 30% of genes that affect the viability of homozygous mutants when deleted (Dickinson et al., 2016). For example, embryonic lethality caused by the deletion of the coding regions of Mixl1 (Pulina et al., 2014), Erbb4 (Gassmann et al., 1995) or Brca1 (Xu et al., 1999) can be rescued by conditional mutagenesis. This generates models that can be used to investigate specific gene-dependent processes during mammalian embryogenesis (Pulina et al., 2014), neurodevelopment (Golub et al., 2004) and breast cancer (Shakya et al., 2008) when combined with an appropriate Cre-expressing line that enables tissue- or developmental-stage-specific gene deletion (Dubois et al., 2006).

The versatility of naturally occurring recombinase-enzyme–target-sequence systems, such as Cre/loxP (Box 1) and Flp/FRT, which derive from bacteria and yeast, respectively, have been adapted to create tools for manipulating mammalian genomes (Gu et al., 1994Rajewsky et al., 1996Dymecki, 1996). These tools have dramatically expanded the types and varieties of alleles that can be designed to study gene function in vivo (Dymecki, 1996Nagy, 2000Nern et al., 2011). A fundamental principle of conditional mutagenesis is the ability to efficiently and reliably convert a functional allele into a mutant one in a specific cell type (called tissue-specific conditional mutagenesis) and/or at a specific time point during development (called time-specific or ‘inducible’ conditional mutagenesis).

Numerous strategies using recombinase-enzyme–target-sequence systems have been developed for conditional mutagenesis (Marth, 1996). Common to all these strategies is the use of short palindromic recombinase target sequences to flank a specific region of a gene (e.g. a critical coding exon common to all transcripts). Such sequences include the Creassociated loxP sequence (to generate a ‘floxed’ allele) or the Flp-associated FRT sequence (to generate an ‘FRT’-flanked allele) (Bouabe and Okkenhaug, 2013). In the absence of the associated recombinase enzyme, these flanking sequences have no effect on normal transcription nor on the expression of the endogenous gene. However, when exposed to the recombinase, the flanking recombinase target sequences recombine with each other to excise or invert the critical coding exon, depending on their orientation and positioning (McLellan et al., 2017) (Poster panel 2A). In its simplest use, if two flanking recombinase target sequences are placed in an asymmetrical head-to-tail orientation, they will recombine to delete the intervening genetic sequence upon exposure to recombinase. Alternatively, if pairs of target sequences are positioned symmetrically in a head-to-head orientation, their recombination will invert the intervening sequence. If target sequences are located on different chromosomes, recombination results in a chromosomal translocation.

There are different ways to elicit recombination. For example, as shown in Poster panel 2B, when a mouse that expresses a floxed allele is mated with a transgenic mouse that expresses the recombinase gene, its progeny will express the recombined allele (Gu et al., 1994). The tissue(s) in which the allele is recombined will depend on the expression pattern of the recombinase, i.e. where the promoter is activated to drive tissue-specific expression of the recombinase. Recombination can also be induced by the in vitro treatment of embryos or tissues with cell-permeable recombinase protein, or via the delivery of viral vectors that express the recombinase (Chambers et al., 2007Lewandoski et al., 1997Su et al., 2002). Recombinase activity can also be targeted to particular tissues by driving the expression of a recombinase from a cell-specific promoter. Recombinase expression can also be induced by expressing the recombinase from an inducible (e.g. drug-responsive) promoter (Sauer, 1998).

The simplest example of the recombinase-enzyme–target-sequence system is shown in Poster panel 2C. This panel shows a molecular targeting construct in which the critical coding exon is flanked by loxP sites. The construct also contains a contiguous endogenous coding sequence of between 3 and 8 kb that is homologous to the wild-type allele. This construct is then introduced into ES cells, for example by electroporation, where it then replaces, via homologous recombination, the endogenous wild-type allele (Hadjantonakis et al., 2008). The conditional allele can then undergo recombination upon exposure to the recombinase to delete the intervening critical coding exon, thereby inhibiting gene expression (null allele).

Another strategy, termed ‘knockout-first’, uses a variation of gene targeting to create a highly versatile allele that combines both gene trap (Friedel and Soriano, 2010) and conditional gene targeting (Jovicić et al., 1990) to generate a lacZ-tagged knockout allele (Testa et al., 2004) (Poster panel 2D). The ‘knockout-first’ allele is generated by inserting an FRT-flanked gene-trap vector, which contains a splice-acceptor sequence upstream of a lacZ reporter gene and a strong polyadenylation stop sequence, into an upstream intron. This creates an in-frame fusion transcript that will disrupt the expression of the targeted allele. Additionally, an adjacent exon coding sequence is flanked with loxP sites (Rosen et al., 2015). This allele can then be converted into a null allele by Cre to abrogate gene expression or into a conditional allele by Flp, which can subsequently be converted by Cre into a null allele (Testa et al., 2004Skarnes et al., 2011). The knockout-first strategy is versatile because it uses a single targeting vector to monitor gene expression using lacZ and tissue-specific gene function using Cre, thereby avoiding embryonic lethality. This strategy has been used effectively to enable the rapid and high-throughput production of thousands of gene knockouts in mouse ES cells in large-scale, genome-wide targeted mutagenesis programs, such as the International Knockout Mouse Consortium (IKMC) (Bradley et al., 2012). Hundreds of mutant mouse models of human genetic diseases have been generated using the knockout-first strategy, including models of skin abnormalities (Liakath-Ali et al., 2014), bone and cartilage disease (Freudenthal et al., 2016), and age-related hearing loss (Kane et al., 2012).

Lastly, an elegant technique termed ‘conditionals by inversion’ (COIN) employs an inverted COIN module that contains a reporter gene (e.g. lacZ) flanked by mutant recombinase target sites (lox66 and lox71) positioned in a head-to-head orientation to enable inversion by Cre recombinase (Albert et al., 1995) inserted into the anti-sense strand of a target gene (Economides et al., 2013) (Poster panel 2E). Cre ‘flips’ the COIN module into the sense strand, interfering with and inhibiting target-gene transcription while activating the reporter. The COIN approach is particularly applicable to single-exon genes and to genes in which the exon–intron structure is not clearly defined. This approach has been used to model an angiogenesis defect in delta-like 4 (Dll4) knockout mice (Billiard et al., 2012) and to generate immunological phenotypes in interleukin 2 receptor, gamma chain (Il2rg) knockout mice (Economides et al., 2013).

Gene expression knockdown using RNAi

About two decades ago, researchers observed that the introduction of double-stranded RNA (dsRNA) that was homologous to a specific gene resulted in its posttranscriptional silencing (Fire et al., 1998). This dsRNA-induced gene silencing was termed RNA interference (RNAi), and it occurs via two main steps (Poster panel 3A). First, Dicer, an enzyme of the RNase III family of nucleases, processes the dsRNA into small double-stranded fragments termed siRNAs (small interfering RNAs; Box 1). Then, the siRNAs are incorporated into a nuclease complex called RISC (for RNA-induced silencing complex), which unwinds the siRNA and finds homologous target mRNAs using the siRNA sequence as a guide; this complex then cleaves the target mRNAs. In the early 2000s, some groups explored whether RNAi could be used to reduce (or ‘knock down’) gene expression in mice by creating transgenic mice that express siRNA (Poster panel 3B). The first proof-of-principle for gene knockdown was demonstrated by delivering lentivirus particles expressing siRNA into green fluorescent protein (GFP) transgenic mice to knock down GFP (Tiscornia et al., 2003). Subsequently, knockdown mice were generated using standard pronuclear injection of constructs that express short-hairpin RNAs (shRNA; Box 1) (Chang et al., 2004Peng et al., 2006Seibler et al., 2007Dickins et al., 2007). Some examples of transgenic knockdown disease models include: an Abca1-deficient mouse line that mimics Tangier disease (Chang et al., 2004); insulin receptor (Insr)-knockdown mice that develop severe hyperglycemia within 7 days (Seibler et al., 2007); and the reversible knockdown of Trp53 as a model useful for tumor regression studies (Dickins et al., 2007).

The advantage of the RNAi knockdown strategy over traditional methods for generating knockout mice is that it provides a rapid and inexpensive approach by which to selectively and, in some cases, reversibly block the translation of a transcript. Although knockdown models can be generated more quickly and cheaply than gene-targeted knockout models (Liu, 2013), a key disadvantage of a knockdown is that transcript inhibition can be variable and transient, and therefore less reliable and reproducible than a knockout. The effects of random insertion, together with varying levels of RNAi in different cells within a tissue, were among the most common pitfalls associated with using RNAi technology to modify mouse gene expression (Peng et al., 2006Yamamoto-Hino and Goto, 2013).

Because of such challenges, and due to the lack of success in generating reliable transgenic RNAi models, this approach did not gain the expected popularity. Alternative strategies were developed to overcome the effect of randomly inserted RNAi constructs by targeting the knockdown cassettes to safe-harbor sites (Box 1), such as the Gt(ROSA)26Sor locus (Kleinhammer et al., 2010) or the Cola1 locus (Premsrirut et al., 2011). These strategies also include making the system modular by incorporating features such as: (i) the Flp-FRT recombinase-mediated cassette exchange (RMCE; Box 1), which facilitates the insertion of a single-copy expression cassette; (ii) a fluorescence reporter that enables gene expression analysis; (iii) microRNA (miRNA) architectures, such as miR30 with reduced general toxicity (McBride et al., 2008); and (iv) tetracycline-inducible elements to enable the expression of the RNAi cassettes upon doxycycline administration (Chang et al., 2004Seibler et al., 2007). A few models that are useful for cancer research have been generated using these approaches, such as Pax5 and eIF4F knockdown models for leukemia (Lin et al., 2012Liu et al., 2014). However, interest in generating knockdown models, as well as in using ES-cell-based gene targeting, began to wane with the development of programmable nuclease technologies (as discussed later).

More recently, an elegant approach that combines the use of the RNA-guided Cas9 nuclease system with RNAi technology has been developed to generate knockdown mouse models by inserting the knockdown cassettes into the intronic sites of endogenous genes (Miura et al., 2015). With this method, a single-copy artificial miRNA against the Otx2 gene was inserted into intron 6 of the Eef2 gene to knock down Otx2 in mid-gestation mouse embryos. This strategy was also used to conditionally activate knockdown cassettes using unidirectional recombinase-mediated inversion of the shRNA cassette. The Miura et al. method offers a feasible and simple strategy to generate gene knockdown models because: (i) it uses an endogenous promoter, unlike other knockdown approaches that require an exogenous promoter to drive the RNAi cassette; (ii) the knockdown cassette is inserted as a single copy at a known site in the genome, unlike approaches that randomly insert the cassette with no control over the number of copies inserted or the number of genomic insertion sites; and (iii) the transgene is not susceptible to silencing, in contrast to other transgenes that are often silenced following random genomic integration.

Pronuclear injection-based transgenesis

Traditional transgenic methods developed over three decades ago involve the injection of linearized DNA expression cassettes into fertilized zygotes (Gordon et al., 1980Palmiter et al., 1982) (Poster panel 4A). Some of the most commonly used transgenic DNA expression cassettes include: (i) cDNA encoding the wild-type or mutant allele; (ii) inducible reporter cassettes, such as the loxP-stop-loxP reporter (Box 1), that incorporate markers such as lacZ or the fluorescent reporters GFP, red fluorescent protein (RFP) or tdTomato; (iii) recombinases, such as Cre (Gu et al., 1994), tamoxifen-inducible Cre (CreERT2) (Feil et al., 1996) and Flp (Dymecki, 1996); and (iv) transcriptional inducers, such as tetracycline transactivators (tTA) or reverse tetracycline transactivators (rtTA) (Gossen and Bujard, 1992).

To produce transgenic mice, a DNA construct is microinjected into the pronuclei (Box 1) of one-cell-stage zygotes (Bockamp et al., 2008). All or part of the injected DNA then inserts randomly at one or more genomic loci as either a single or as multiple (e.g. tandem-repeat) copies. The suitability of this approach for generating animal models is limited by the uncertainty of obtaining a desired level of gene expression due to the random nature of transgene insertion and copy number (Chiang et al., 2012). As a result, ES-cell-based methods were developed to target expression cassettes (such as those encoding Cre) into a specific locus in the genome; for example, the Gt(ROSA)26Sor locus, which enables the ubiquitous expression of an inserted transgene (Soriano, 1999). Depending on the construct and insertion site, transgene expression could be driven by a target gene’s endogenous promoter and/or by other regulatory elements (Rickert et al., 1997). In this way, an intact, single-copy transgene becomes integrated into a predetermined genomic location in ES cells via homologous recombination, thereby optimizing transgene expression (Rickert et al., 1997Soriano, 1999). The targeted ES cells are then introduced into morulae or blastocysts, as previously explained, before being implanted into pseudopregnant females. Although this approach overcomes some of the constraints inherent to random transgenesis (such as high variability of gene expression, and difficulty in obtaining the desired transgene expression patterns and levels), homologous recombination has technical hurdles of its own that make it expensive, labor intensive and time consuming. In addition, germline transmission of the exogenous allele can fail, creating a frustrating struggle for researchers who need to reliably and regularly manipulate the mouse genome (Ohtsuka et al., 2012a). Another disadvantage of the ES cell targeting approach is that ES cell genomes do not always remain stable in culture, and can undergo changes before and after gene targeting (Liang et al., 2008).

The recently developed targeted transgenic technologies enable the integration of single-copy transgenes at specific loci in the genome, directly via pronuclear injection. In pioneering work, Masato Ohtsuka and co-workers developed a method called pronuclear injection-based targeted transgenesis (PITT) (Ohtsuka et al., 2010), which allows a single copy of a complete transgene to be precisely inserted at a desired genomic locus in the zygote (Poster panel 4B). The PITT method involves two steps. First, a landing pad (for example, a cassette containing a combination of mutant loxP sites) is inserted at a defined locus in ES cells to generate a ‘seed’ mouse strain. Second, the PITT components – a donor plasmid containing the DNA of interest (DOI) and a Cre source (either plasmid or mRNA) – are injected into fertilized eggs collected from the seed strain mice. The DOI inserts at the landing pad via recombination-mediated cassette exchange (RMCE). The landing pad and the donor DNA contain compatible sequence elements that enable the donor DNA to insert precisely into the target locus. In the first report (Ohtsuka et al., 2010), the authors employed a well-established CreloxP system (as the components of the landing pad and the donor plasmid elements) to achieve RMCE. Soon after the first description of the PITT technology, another group reported a similar approach using the PhiC31 integrase and attP/B system, which correspond to the landing pad components and donor plasmid elements (Tasic et al., 2011). This modified method to achieve targeted transgenesis was named Targatt™ (Chen-Tsai et al., 2014). The main advantages of the various targeted transgenesis methods that use either CreloxP recombination or PhiC31attP/B integration, are that: (i) they overcome the problems associated with random transgene insertion, such as fragmented insertion of the transgenes, multicopy insertions, transgene silencing or interference in the expression of the endogenously disrupted gene; and (ii) they resolve the time and cost limitations associated with ES-cell-based approaches by targeting DNA cassettes to specific sites in the genome.

In initial reports of the PITT method, the Cre recombinase was encoded by a plasmid, and the plasmid DNA was injected into the pronuclei of zygotes together with the donor DNA. This method has since been improved by: (i) the use of Cre mRNA instead of plasmid DNA, which was done because plasmid DNA needs to be transcribed, which delays the expression of Cre, by which time the donor DNA might have degraded (Ohtsuka et al., 2012b); (ii) the development of new PITT-compatible donor vectors (Ohtsuka et al., 2012b); and (iii) the development of a seed mouse strain that contains both CreloxP and PhiC31-attP/B cassette insertion systems, providing researchers with the flexibility to use either (Ohtsuka et al., 2015). In this format, multiple different PITT donor plasmids can be included in the microinjection mix: any one of these donors can be inserted at the landing pad in separate founder mice, resulting in independent transgenic mouse lines generated in a single session of microinjection. These latest technical tools, dubbed ‘improved PITT’ (i-PITT), allow up to three transgenic mouse lines to be generated simultaneously, such that each line has a different DOI after a single microinjection session (Ohtsuka et al., 2015). The PITT technology is reviewed in detail in Ohtsuka et al., 2012a and a comprehensive list of available PITT tools was recently described (Schilit et al., 2016). The PITT/i-PITT approaches have been used to generate many reliable single-copy transgenic reporter mouse lines that are useful for disease research, including in neuroscience (Madisen et al., 2015) and nephrology (Tsuchida et al., 2016). For example, Tsuchida et al. (2016) reported generating a nephrin-promoter-driven EGFP transgenic mouse model; they further showed that cultured glomeruli from this model serve as tools to screen for compounds that enhance nephrin-promoter activity. Although PITT strategies have overcome the limitations of random transgenesis, a major pitfall of this approach is that custom PITT seed mouse strains need to be generated for a given locus and maintained as breeder colonies as zygote donors for targeted transgenesis.

Despite the technical advances in genetic engineering over the past four decades, one recent and remarkable technical breakthrough is rapidly superseding nearly all of these advances: programmable endonucleases.

Programmable endonucleases for genome editing

Programmable endonucleases bypass the classical ES-cell-based gene-targeting steps to engineer a precise and heritable mutation at a specific site in the genome. Injection directly into one- or two-cell-stage embryos enables the germline modification of a specific genetic locus without the need for the three complex steps above.

Programmable endonucleases can introduce genetic mutations in one of two ways (Joung and Sander, 2012Gaj et al., 2013Sander and Joung, 2014Cox et al., 2015). They can cause: (i) imprecise, error-prone DNA repair as a result of non-homologous end joining (NHEJ; Box 1) of the cleaved DNA ends; or (ii) the precise repair of cleaved DNA ends by homology-directed repair (HDR; Box 1) via the co-injection of a DNA repair template. Nonetheless, the imprecise insertion of the donor DNA can still occur in HDR-mediated repair. The development of programmable endonucleases for genome editing has opened up a whole new set of technical possibilities to create animal models for biomedical research using virtually any suitable species.

There are four major platforms that employ programmable endonucleases, which were initially discovered in microbiology research applications (Chevalier and Stoddard, 2001Li et al., 1992Mojica and Garrett, 2013Mojica et al., 1993Römer et al., 2007) and have since been repurposed for editing the genomes of higher animals, including mice. They are, in the order they were developed: homing endonucleases (HEs); zinc-finger nucleases (ZFNs); transcription activator-like effector nucleases (TALENs); and the clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) system (Poster panel 5). Common to all four programmable endonuclease platforms is their sequence-specific nuclease activity, which allows researchers to cleave DNA at a specific target site for genome editing (Joung and Sander, 2012Gaj et al., 2013Sander and Joung, 2014Cox et al., 2015).

The HEs were among the first of the endonucleases (Rouet et al., 1994) to be used for genome manipulation. Although HEs were shown to increase gene-targeting efficiency in ES cells (Smih et al., 1995), there is little evidence to suggest that they have been used successfully to genetically engineer mutant mice. This is probably because of the numerous steps required to design and construct HEs to target specific genomic sites, and because only a small number of genomic sites could be targeted. The ZFNs, unlike HEs, offered greater flexibility as they are easier to engineer and can target more genomic locations than can HEs (Poster panel 5). From 2002 onwards, ZFNs became more widely used than HEs, especially as a research tool in various organisms, including flies, fish and plants (Urnov et al., 2010Carroll, 2011). The first ZFN-modified mutant mouse models were described in 2010 by Carbery and co-workers via the direct injection of ZFNs that target and inactivate Mdr1aJag1 and Notch3 (Carbery et al., 2010). Nevertheless, the technical complexity of building ZFNs, and intellectual property restrictions, limited their widespread adaptability. TALENs, the next set of programmable nucleases, were developed in 2010 and overcame many of the limitations of HEs and ZFNs. TALENs were simpler, easier to build and could be used to target a greater number of genomic sites than could HEs or ZFNs, and thus were immediately adopted by hundreds of labs as research tools. The first mutant mouse models using TALENs were developed by Sung and co-workers in 2013 via the direct injection of TALENs that targeted Pibf1 and Sepw1 to inactivate them (Sung et al., 2013).

At the time when ZFNs and TALENs were being developed, each platform proved to be quite versatile and superior to the previously available genetic engineering tools. Then came the development of the CRISPR/Cas9 genome editing tool in late 2012 and early 2013 (Jinek et al., 2012Cong et al., 2013Mali et al., 2013) (Poster panel 5). A series of papers from multiple groups, published within a few months of each other, demonstrated that dsDNA breaks at specific sites in the genome could be generated with very high efficiency in mammalian cells by using guide RNAs complementary to the target site and the Cas9 nuclease (Jinek et al., 20122013Mali et al., 2013Cong et al., 2013Cho et al., 2013). Within just a few months, some groups demonstrated that the RNA-guided Cas9 nuclease system could be used to rapidly generate mutant mouse models (Shen et al., 2013Wang et al., 2013). Since then, the RNA-guided Cas9 nuclease system has almost completely superseded all other technologies for genome editing. A direct comparison of the RNA-guided Cas9 nuclease system with the previous nuclease-based platforms (HEs, ZFNs and TALENs) clearly shows that it has several advantages (Sander and Joung, 2014Porteus, 2015Woolf et al., 2017). These include its simplicity of use, lower cost and higher efficiency. The RNA-guided Cas9 nuclease system is constantly being improved to make it increasingly efficient and versatile, including optimizing and improving the efficiency of existing Cas nucleases (Kleinstiver et al., 2016Slaymaker et al., 2016), and the development of novel Cas nucleases (Shmakov et al., 2015Zetsche et al., 2015). The RNA-guided Cas9 nuclease system is considered a ‘disruptive’ technology because it is quickly making previously well-established and fully developed technologies outdated. In recent years, researchers have come to prefer this approach over ES-cell-based gene-targeting methods (Burgio, 2018Skarnes, 2015) because RNA-guided Cas9 nuclease approaches are relatively quicker, less expensive and less cumbersome.

The versatility of the RNA-guided Cas9 nuclease system allows researchers to engineer and edit the genome in ways that were previously not possible using non-nuclease-based approaches (Poster panel 5). This includes the ease and speed with which researchers can induce a footprint-free point mutation (Box 1) (Inui et al., 2014Gurumurthy et al., 2016a). Many human disease conditions are caused by subtle genetic changes, such as point mutations, or by the addition or deletion of a few nucleotides (Gonzaga-Jauregui et al., 2012). Developing animal models of such subtle genetic changes, by using ES-cell-based targeting approaches, inevitably requires the addition of other genetic elements near the vicinity of the genetic change [such as a drug selection marker (neomycin or puromycin) and recombinase elements (such as loxP or FRT sites)]. By contrast, the RNA-guided Cas9 nuclease system can generate animal models with subtle genetic changes with high precision, rapidly, efficiently and without leaving any residual genetic alterations. Compared to previous methods, this capability represents a significant advance in murine genome editing for human disease modeling. The RNA-guided Cas9 nuclease tool has also facilitated the generation of multiple mutant mouse models in a single experiment by inducing dsDNA breaks at multiple target sites, resulting in several different gene disruption models (Wang et al., 2013). The RNA-guided Cas9 nuclease system also enables the generation of mutant mouse models on genetic backgrounds that were not amenable to being genetically manipulated with earlier approaches, such as the immunodeficient NOD/Scid-ILgamma (NSG) strain (Li et al., 2014). The RNA-guided Cas9 nuclease system has also become a powerful tool for both forward and reverse genetics (Gurumurthy et al., 2016c), generating models that are relevant for many diseases, including cancer (Platt et al., 2014). Several recent review articles discuss the Cas9-nuclease-generated mouse models for different disease types, including for cancer (Mou et al., 2015Roper et al., 2017), cardiovascular diseases (Miano et al., 2016), neurodegenerative diseases (Yang et al., 2016) and kidney diseases (Higashijima et al., 2017). In addition, several reviews on Cas9-nuclease-generated models have been recently published that discuss their human disease relevance (Dow, 2015Tschaharganeh et al., 2016Cai et al., 2016Yang et al., 2016Birling et al., 2017).

Despite its advantages, the RNA-guided Cas9 nuclease system poses challenges, such as mosaicism (Yen et al., 2014) and off-target effects. If one of the two haploid genomes in the one-cell-stage zygote is not cleaved before the zygote divides, or if Cas9 activity persists at the two-cell or later stages, additional mutant alleles can be generated, resulting in more than three mutant alleles in the developing offspring. Consequently, as many as six or more types of alleles were detected in one founder (G0) mouse (Li et al., 2013). It is therefore essential to genotype F1 offspring to identify a desired mutant allele. This mosaicism can also be considered an advantage because multiple different alleles can be segregated and used as separate mutant models. For example, the same founder mouse could contain a complete insertion deletion (indel) allele and the foreign cassette knock-in allele; each can be used for different research applications. Because the Cas9 target sequence is only 23 nucleotides long, including the protospacer adjacent motif, it is likely that imperfect target-matching sequences are present elsewhere in the genome that contain one or a few mismatches. Cas9 can potentially bind to such imperfect target sites and thus generate dsDNA breaks and indels at those sites. Indel mutations in off-target sites can have confounding effects in mouse phenotyping experiments. However, off-target effects are not considered a major concern because they: (i) are generally negligible in mice (Iyer et al., 2015); and (ii) can be segregated during mouse breeding. Another recent study, now retracted, reported the presence of high rates of off-target effects in Cas9 engineered mice (Schaefer et al., 2017); however, this report’s experimental design and interpretations have been questioned by the scientific community (Kim et al., 2018Lescarbeau et al., 2018Nutter et al., 2018Wilson et al., 2018).

A current challenge to the broader use of RNA-guided Cas9 nuclease is the inability to use it to insert large fragments of DNA reliably and efficiently. Because most genetic-engineering approaches in mice involve the insertion of engineered DNA cassettes, efforts are underway to improve the ‘knock-in’ capabilities of this system. While a few RNA-guided Cas9 nuclease strategies have been modified to support the insertion of new cassettes (Aida et al., 2015Maruyama et al., 2015Sakuma et al., 2016), including a strategy that combines PITT and RNA-guided Cas9 nuclease approaches (Quadros et al., 2015), none has yet been successfully adapted for the routine engineering of the mouse genome. A report from Ohtsuka’s group, which used long single-stranded DNA (lssDNA) donors (generated via in vitro transcription and reverse transcription), demonstrated that lssDNAs could serve as efficient donors for insertion at the Cas9 cleavage sites (Miura et al., 2015). Another report, which used lssDNAs purified from nicked plasmids to create rat knock-in models, also demonstrated that the lssDNA donor strategy could be a reliable approach for creating insertion alleles (Yoshimi et al., 2016). More recent reports show that co-injecting lssDNA donors with commercially available CRISPR ribonucleoprotein complexes (instead of the previous formats of Cas9 mRNA and sgRNAs), offers a highly robust and efficient strategy for insertion alleles in a method termed Easi-CRISPR (efficient additions with ssDNA inserts-CRISPR) (Quadros et al., 2017Miura et al., 2017).

RNA-guided Cas9 nuclease reagents have also been delivered into zygotes via electroporation of RNA and/or of ribonucleoproteins (Chen et al., 2016Hashimoto and Takemoto, 2015Qin et al., 2015). The ability to deliver RNA-guided Cas9-nuclease gene-editing reagents into several zygotes at once overcomes the need to inject each individual zygote, one at a time, and greatly simplifies the process of generating mouse models. Furthermore, electroporation is less damaging to embryos than microinjection (Chen et al., 2016Hashimoto and Takemoto, 2015Qin et al., 2015). Another advance in delivering the RNA-guided Cas9 nuclease system is a method called GONAD (genome editing via oviductal nucleic acids delivery). This procedure delivers Cas9 reagents to embryos in the oviduct using electroporation (Takahashi et al., 2015Gurumurthy et al., 2016bSato et al., 2016Ohtsuka et al., 2018). Unlike standard approaches, this method does not require any of the three major steps of animal transgenesis: zygote isolation from a female donor; ex vivo handling of zygotes (involving either microinjection or electroporation); and the transfer of zygotes to a pseudopregnant female mouse. This approach requires surgical skills that are equivalent to performing the oviductal transfer of embryos. The GONAD method can be used to generate knockout mice (Takahashi et al., 2015), and, by using the so-called improved-GONAD (i-GONAD), more complex animal models, such as knock-ins and large-deletion models, can be generated at an efficiency similar to the microinjection-based methods (Ohtsuka et al., 2018). The i-GONAD method also uses only a third of the mice used in standard microinjection or in ex vivo zygote electroporation methods (Ohtsuka et al., 2018). These methods need not be limited to centralized facilities, sophisticated equipment or highly skilled technical personnel. It is thought that the technical advances such as Easi-CRISPR and i-GONAD have the potential to entirely reshape the traditional route of generating modified alleles in mice if the techniques are widely adopted by many research groups and by transgenic core facilities (Burgio, 2018).

Concluding remarks and future perspectives

Recent technological breakthroughs have enabled very rapid changes in the way we generate genetically altered mouse models. Most notably, the RNA-guided Cas9 nuclease system is assuming a key role in shaping this new technological landscape. While the use of the RNA-guided Cas9 nuclease system has transformed and eclipsed traditional transgenic technologies in many ways, challenges remain, including the inability to insert large DNA constructs to generate a knock-in mouse (Box 1) with reporter, conditional or humanized alleles, or to engineer chromosomal rearrangements and other complex alleles easily, routinely and efficiently.

Genetic manipulation also underpins the ongoing efforts to elucidate the functional roles of every gene in the mouse genome, as a first step to understanding the role of ‘disease alleles’ identified by the exome and genome sequencing of human patients. Genomic and precision medicine depends on our ability to differentiate benign from pathogenic variant alleles, and disease-causing alleles from the longer list of disease-associated ones. Genetic manipulation of the mouse genome is thus essential for understanding gene function and for uncovering the genetic and molecular basis of human disease, leading to improved diagnostic accuracy, development of targeted therapeutics and the implementation of effective prevention strategies.

Footnotes

  • © 2019. Published by The Company of Biologists Ltd

http://creativecommons.org/licenses/by/4.0

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Mouse Model

Mouse models can be considered as the starting point for investigating a certain basic principle, without having an aim to translate it into humans, just like other non-human models such as yeast, fruit fly or zebrafish, depending on the nature of the question in study: for example, studying the mechanism of heat shock proteins in the antigen presentation process or understanding the survival mechanism of the gut nematode against the host immune response.

From: The Laboratory Mouse (Second Edition), 2012

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Mouse Models

Siân E. Piret, Rajesh V. Thakker, in Genetics of Bone Biology and Skeletal Disease, 2013

II Methods for Generating Mouse Models

Non-Targeted Strategies

Spontaneous mutations in mice may result in benign phenotypes such as variable coat colors, or in disorders that have similarities to diseases in humans, e.g. the hypophosphatamia (Hyp) mouse, which is representative of X-linked hypophosphatemia in humans.52 Such spontaneous mutations occur at very low frequencies, thus several techniques that increase the rate of mutation induction in the mouse genome by either non-targeted (random) or targeted strategies have been developed (see Tables 13.1 and 13.2). An early example of non-targeted mutagenesis is provided by irradiation, which generated the Gy mouse, a second model for X-linked hypophosphatemia.52 More recently, chemical mutagens have been used in large-scale mutagenesis programs. Successful agents include isopropyl methane sulfonate (iPMS) used to generate the Nuf mouse model with an activating calcium-sensing receptor (CaSR) mutation, and N-ethyl-N-nitrosourea (ENU) used to generate a mouse model for osteogenesis imperfecta with a collagen 1 alpha 1 (COL1A1) mutation. ENU, which is an alkylating agent that primarily introduces point mutations via transfer of the ENU alkyl group to the DNA base followed by mispairing and subsequent basepair substitution during the next round of DNA replication (Figure 13.1A), is the most potent mutagen in mice.14 Intraperitoneal injections of ENU to male mice generate approximately one mutation per 1–1.5 Mbp of sperm DNA,14 which allows the mutations to be inherited (Figure 13.1B). ENU mutagenesis programs utilize two complementary approaches, which are phenotype-driven and genotype-driven screens. In phenotype-driven screens, the offspring of mutagenized mice are assessed for phenotypic variances, using a panel of morphological, biochemical, or behavioral tests, in a “hypothesis-generating” strategy, which aims to elucidate new genes, pathways and mechanisms for a disease phenotype14 (Figure 13.1B). By establishing appropriate matings, phenotype-driven screens can be used to identify dominant or recessive phenotypes. Genotype-driven screens, in which mutations in a gene of interest are sought, are “hypothesis-driven” and are feasible by using available parallel archives of DNA and sperm samples from mutagenized male mice (Figure 13.1B). Archived DNA samples from the mutagenized male mice are used to search for mutations in the gene of interest, and once mutations are identified in the mouse DNA, then the corresponding sperm sample for the male mouse harboring the mutation is used to establish progeny carrying the mutation by in vitro fertilization.14 It is estimated that the probability of finding three or more mutant alleles in an archive of >5000 DNA samples is >90%.53 Thus, the gene-driven approach can be used to generate an “allelic series” of mutations within one gene, which may yield insights into genotype–phenotype correlations in the gene and disease of interest.54

ENU mutations most frequently result in missense mutations (>80%) that may generate hypo- and hypermorphs, although occasionally nonsense or frame-shift mutations (<10%) generating knockout models may be obtained.55 However, a more recent and reliable method for generating non-targeted knockout models on a large scale is by the use of insertional mutagenesis, utilizing gene-trap strategies.56,57 Gene-trap vectors usually consist of a reporter gene, either with or without a promoter, and a strong splice acceptor site, which causes any upstream exons to splice directly to the gene-trap15 (see Figure 13.1C). The vector is either electroporated or retrovirally infected into embryonic stem (ES) cells, after which it randomly inserts into the genome. Mutagenized ES cells are then re-introduced into developing blastocysts to generate chimeric mice, from which germline mutant mice can be bred (Figure 13.2). A recent refinement of the gene-trap strategy is targeted trapping, in which the vector also contains regions homologous to the targeted gene, thereby facilitating the deletion of a specific gene.16,56

Targeted Strategies

A specific loss of function (i.e. knockout) of a gene of interest in the germline can be generated to yield conventional targeted knockout models, as follows. A targeting construct is assembled, which contains two “arms” of sequence homologous to the gene of interest and that flank a positive selection cassette such as the E. coli neomycin phosphotransferase (NeoR) gene (Figure 13.3A). Integration of the NeoR gene (and therefore the targeting construct) into the ES cell genome allows these ES cells to survive normally toxic amounts of antibiotic treatment, thereby allowing selection of ES cells that have been successfully targeted by homologous recombination. Furthermore, replacement of an exon (or exons) by the NeoR cassette results in gene disruption, i.e. “knockout”17 (Figure 13.3A). To facilitate further the selection of ES cells that have undergone successful targeting by homologous recombination, a negative selection cassette, such as the Herpes simplex virus thymidine kinase (TK) gene, may also be used. The TK gene cassette is inserted at one end of the homologous region of the targeting construct, such that the TK cassette is lost if homologous recombination has occurred (Figure 13.3A), but retained if non-homologous recombination has occurred. In the presence of a thymidine analog in the growth medium, ES cells containing the TK cassette (i.e. following non-homologous recombination) will not undergo cell division, as the thymidine analog will undergo phosphorylation and will be incorporated into the DNA by the TK, and thereby disrupt cell division, hence selecting out these ES cells. In contrast, those ES cells that do not have the TK cassette (i.e. following homologous recombination) will not have disrupted cell division due to incorporation of the thymidine analog and, as a result, will proliferate.17 Correctly targeted ES cells are used to generate chimeric mice (see Figure 13.2), which are then bred with wild-type mice to yield mice with germline transmission of the disrupted allele, i.e. “knockout” mice, that have one copy of the disrupted allele in all of their cells. Cross-breeding of these heterozygous knockout mice can then yield homozygous knockout mice, which will have a disruption of both alleles of the gene in all of their cells. These “conventional” knockout models have proved to be very useful in studies of human diseases, although their use may be limited if the disruption of the gene in a critical organ results in early death, e.g. at any embryonic stage. To overcome such limitations, it may be useful to generate tissue-specific (i.e. conditional knockout) or time-specific (i.e. inducible knockout) models. This can be achieved by refining the gene-trap and “conventional” knockout strategies by the addition of either LoxP or flippase (FLP) recombinase target (FRT) sites in the targeting vector (Figure 13.3B). LoxP and FRT sites are short DNA sequences which are recognized and acted upon by the enzymes Cre (causes recombination) recombinase or FLP recombinase enzymes, respectively and, when inserted to flank the genomic region of interest, will result in either excision or inversion of the DNA flanked by the LoxP or FRT sequences, depending on whether the two sequences are in the same orientation (Figure 13.3B), or opposite orientations, respectively. Thus, insertion of the LoxP and FRT sequences allows several variations on the knockout mouse, including either tissue-specific (conditional) or time-specific (inducible) knockouts (see Table 13.2). Thus, if mice containing alleles in which the exon containing the start codon is flanked by LoxP sites (“floxed”) or FRT sites (“flirted”), are crossed with transgenic mice expressing Cre or FLP under the control of tissue-specific promoters (e.g. the PTH gene promoter for parathyroid-specific expression), the gene of interest can be knocked out in a specific tissue (Figure 13.3B). The inducible models utilize a fusion protein, such as a modified ligand-binding domain of the estrogen receptor fused to the Cre (CreER) or FLP gene which, on administration of an estrogen receptor antagonist (tamoxifen), translocates to the nucleus to excise the floxed allele(s), thereby allowing the gene to be permanently knocked out at the desired time, which may be either during embryonic or neonatal development, or in adult life.18 These conditional and inducible knockout mouse models have proved particularly useful to overcome embryonic or early postnatal lethality, for example in studies of Blomstrand’s chondrodysplasia due to PTHR1 loss of function (see Table 13.4), or to understand the role of a protein in one particular tissue.

Knockout mice have been very valuable for the study of physiological functions of proteins and the elucidation of disease mechanisms. However, knockout models are not always the most appropriate, particularly when the human disease being studied is not due to a loss of function or null allele for the gene. Indeed, the majority of human diseases are unlikely to be due to null alleles, but are instead associated with point mutations, which may result in a constitutively active protein, or a toxic gain of function, as illustrated by PTHR1 mutations in Jansen’s disease (see Table 13.4), or dominant negative effects. Thus, to generate appropriate murine models for these diseases, the specific mutation needs to be introduced into the mouse genome, and this may be achieved by targeted knock-in or transgenic approaches (see Tables 13.1 and 13.2). The generation of targeted knock-in models utilizes a similar approach to that described above for targeted knockout models, except that a targeting vector which carries the desired mutation must be specifically generated (see Figure 13.3C). In addition, the positive selection cassette is normally placed in an intron and floxed so that it can be excised and cause minimal effects on gene expression.19 The generation of transgenic models utilizes a targeting construct which usually contains the cDNA carrying the mutation, together with an appropriate promoter and poly(A) sequence, which is injected into the pronucleus of fertilizd mouse eggs.20,21 The transgene undergoes random insertion into the genome, and several copies are often inserted together, which therefore generates an overexpression model. As reviewed below, these different strategies for generating mouse models of human diseases have greatly facilitated studies of inherited bone and mineral disorders that have investigated mechanisms and treatments, which would not be easily feasible in patients.View chapterPurchase book

Mouse Models of The Nuclear Envelopathies and Related Diseases

Henning F. Horn, in Current Topics in Developmental Biology, 2014

9 Conclusions

Mouse models provide a valuable tool for studying human diseases. This has certainly been true for mouse models of LINC complex proteins and their associated diseases. The various Nesprin-1 mouse models have augmented our understanding of the underlying biology of muscular dystrophyautosomal recessive arthrogryposis, and autosomal recessive cerebellar ataxia. The mouse models for the Nesprin-4-associated hearing loss were critical in elucidating the cell biology and provided key insights into this entirely novel class of human hearing loss. And mouse models of SUN1 and KASH5 have allowed us a greater appreciation for the importance of chromosomal movement in the development of gametes. Indeed, our mouse model-generated understanding of LINC complex functions may even prove to be predictive for human diseases. For example, a novel human disorder was recently described that has features of mandibular acral dysplasia but also includes deafness and male hypogonadism as prominent associated features (Shastry et al., 2010). Several candidate genes were examined, but no mutations were found to cause this genetic condition. However, given our knowledge of the roles of SUN1, and the phenotypes of the SUN1 mouse models (hearing loss and hypogonadism), it would be interesting to check the function of SUN1 in these patients.

While a number of mouse models now exist for LINC complex proteins, the field is still relatively young. Indeed, we are still discovering novel LINC complex functions and novel variants of LINC complexes. We therefore expect that future mouse models will continue to augment our understanding of the LINC complex in normal as well as pathophysiological roles.View chapterPurchase book

Alzheimer’s Disease: Transgenic Mouse Models

K.H. Ashe, in Encyclopedia of Neuroscience, 2009

Transgenic mouse models of Alzheimer’s disease (AD) have been created to study the structural and functional consequences of the accumulation of the amyloid-β and tau proteins in the brain. They have also been used to test experimental therapeutic interventions for AD. No transgenic mouse model perfectly represents all stages and facets of AD; transgenic mouse models cannot supplant the need for studying the disease in humans and human clinical trials. However, studies in transgenic mouse models allow researchers to understand aspects of the pathophysiology of AD and coordinate efforts to diagnose and treat the illness in humans.View chapterPurchase book

In Vitro and In Vivo Animal Models

Azka Khan, … Alvina Gul, in Omics Technologies and Bio-Engineering, 2018

18.2.13 Transgenic Mouse of PD

Mouse models have been a vital tool for research in neurodegenerative diseases. They have been proved as an effective model organism for PD. Both in vitro and in vivo mouse models have been extensively used. Many transgenic mouse models have been generated to study PD; α-synuclein protein has very important role in the pathology of this disease. KO mice and some transgenic mice with the ability to overexpress α-synuclein possess familial A53T or A30P mutations. α-Synuclein KO mice are viable and fertile, and they support a significant role of α-synuclein in regulation of dopaminergic neurotransmission, synaptic plasticity, and presynaptic vesicular release and recycling (Janus and Welzl, 2010).View chapterPurchase book

Biomarkers for Assessing Risk of Cancer

Xifeng Wu, Jian Gu, in The Molecular Basis of Cancer (Fourth Edition), 2015

Mouse Models for Cancer Susceptibility Study

Mouse models that cross tumor-resistant with tumor-susceptible strains have been instrumental in mapping several candidate cancer susceptibility loci and expression quantitative trait loci (eQTLs)125-131 before the wide application of GWAS in human cancers. Although hundreds of cancer susceptibility loci have been identified through GWAS, the majority of the heritable risk of cancer cannot be explained by the main effects of common alleles. Gene-gene and gene-environment interaction clearly play important roles in cancer development, which is challenging in human studies because of the heterogeneity of human cancers. Mouse models have a defined genetic background that does not possess the genetic heterogeneity characteristic of human cancers. Crossing genetically distinct mouse strains can allow the analysis of the combinatorial effects of host genetic background and somatic events at different stages of cancer development. A recent study applied a network analysis in a mouse model of skin cancer that produces both benign tumors and malignant carcinomas and identified a genetic architecture affecting inflammation and tumor susceptibility.132 Gene–environment interactions can also be investigated using mouse models to identify how genetic modifiers of tumor initiation interact with specific environmental effects identified through epidemiological studies. Mouse models will also be a major tool for mechanistic studies of cancer susceptibility loci.View chapterPurchase book

Molecular Basis of Lung Cancer

Mitsuo Sato, … John D. Minna, in The Molecular Basis of Cancer (Third Edition), 2008

New Transgenic Mouse Models of Lung Cancer

Mouse models that recapitulate the carcinogenic process of human lung cancer are powerful tools to improve our understanding of lung cancer pathogenesis, develop targeted therapeutics, and evaluate their in vivo efficacies. Several different types of transgenic mouse models for studying lung cancer have been developed with innovative strategies. Bitransgenic models using Cre/LoxP recombination or tetracycline-inducible gene expression system have enabled regulating the expression of a gene in mice in a timely and spatially controlled manner. Two groups engineered mouse strains harboring conditional mutant K-ras alleles that are expressed only after Cre/LoxP-mediated recombination occurs. Both groups showed that oncogenic K-ras activation induces lung adenocarcinoma, demonstrating the contributions of oncogenic K-ras to lung cancer pathogenesis (46). Moreover, Meuwissen et al. developed a mouse model of SCLC by inactivating both Rb and p53 using Cre/LoxP recombination system (46). Using a tetracycline-inducible gene expression system, mice harboring EGFR tyrosine kinase domain mutations were engineered. These mice developed adenocarcinomas very similar to human adenocarcinomas with EGFR mutations. Although there can be significant differences in lung tumor development between humans and mice, mouse models have a complete physiologic environment and allow analyzing host tumor interaction and angiogenesis, which cannot be studied in tissue culture. Finally, no mouse model of squamous cell carcinoma of the lung has been developed.View chapterPurchase book

Huntington Disease

Laura A. Wagner, … Blair R. Leavitt, in Animal and Translational Models for CNS Drug Discovery, 2008

Validity of Animal Models of HD

Mouse models of HD are important to the discovery and validation of drug targets for HD as well as central to proving drug efficacy preceding human therapeutic trials. The development and validation of an effective mouse model of disease is no trivial matter and requires extensive characterization and rigorous validation (see Table 6.2). The ideal mouse model for HD agrees in etiology, pathophysiologysymptomatology, and response to therapeutics when compared to the human condition. Originally, chemical models of HD were investigated based on their similar striatal neurodegenerative pattern as seen in human HD patients. These chemical models met the very basic symptomatology criterion alone. Since the discovery of the HD gene, however, more accurate gene models of HD have been developed as transgenic mice representing HD etiology, pathophysiology, and symptomatology. Although species differences complicate the exact phenotype comparisons that can be made, genetic HD mice overall recapitulate cognitive failure, motor dysfunction, and striatal neurodegeneration as seen in human HD patients.

Table 6.2. Validation of animal models of diseasea

Face validity- a superficial resemblance between the mouse model and human disease. A similarity seen in symptoms is a common justification in this case (e.g., chemical models of HD).
Predictive validity- the ability of a model to predict the performance of the condition being modeled. One example is a model’s capacity to predict compound efficacy in therapeutic human trials.
Construct validity- a theoretical clarification of what a model is supposed to represent. This validation accounts for the inherent difference that may occur in a process when looking across species.
Etiological validity- in this case the model and the human condition must undergo identical etiologies. The simplest disease to model in this situation is that of a simple inheritance disease.

aVan Dam and De Deyn. (2006). Drug discovery in dementia: The role of rodent models. Nat Rev: Drug Discov 5:956–970.

Three basic design strategies have been applied in developing HD gene mouse models giving rise to three broad model categories including: (i) fragment models containing N-terminal fragments of the human mutant Htt protein in addition to both alleles of murine Hdh, (ii) full-length models containing the full-length human HD gene with an expanded polyglutamine tract in addition to both alleles of murine Hdh, and (iii) knock-in models of HD with pathogenic CAG expansions in murine Hdh. Individually these gene models are believed to represent certain aspects of HD based on their design and phenotype. These characteristics help define the strength of the model and its subsequent use in the field of HD research. Together these different gene models provide confirmatory proof of the dysfunction and disease caused by a Htt CAG expansion in mice. As a result, HD gene mouse models provide a powerful analysis for target validation and drug discovery preceding clinical trials. To date, the fourth criterion of an ideal HD mouse model, its predictive power in identifying effective drugs for HD awaits verification by emerging and ongoing human clinical trials.View chapterPurchase book

Application of Mouse Genetics to Human Disease

Teresa M. Gunn, Brenda Canine, in Rosenberg’s Molecular and Genetic Basis of Neurological and Psychiatric Disease (Fifth Edition), 2015

Summary

Mouse models have led – and are certain to continue to lead – to significant breakthroughs in identifying genes, mechanisms, and pathways that underlie human neurologic diseases. Mice are also ideal for testing therapeutic approaches, something we are likely to see more of in the coming years. New methodologies have increased the speed and accuracy with which new mouse models can be generated, and technological advances have led to improved tools to analyze them. Models of multigenic disorders remain scarce. This is primarily because it is difficult to identify the variants that cause these traits, and most mouse models are presently generated using gene targeting, which requires the causative loci be known. Random mutagenesis and thorough phenotypic analysis (including behavioral studies) of existing mutants may reveal subtle and/or unexpected traits, and will complement other, ongoing projects aimed at discovering disease-associated variants in human populations. There is much excitement over the ability to reprogram fibroblasts or other patient-derived cells into induced pluripotent stem cells (iPSC), and the ability to differentiate those iPSC into neuronal stem cells allows for the analysis of those cells in culture. Injecting these cells into the mouse brain will create a new class of mouse models that will provide insight into the in vivo behavior of patient-derived cells in the mammalian nervous system. Combining these models with existing genetic models and reporter mice will create a powerful system for analyzing the pathogenesis of neurological disorders.View chapterPurchase book

Gsα, Pseudohypoparathyroidism, Fibrous Dysplasia, and McCune–Albright Syndrome

Lee S. Weinstein, Michael T. Collins, in Genetics of Bone Biology and Skeletal Disease (Second Edition), 2018

3.4 Animal Models

Mouse models leading to constitutively activation of cAMP formation have been created by transgenic overexpression of Gsα, by expression of R201H or Q227L mutant forms of Gsα, or by expression of the cholera toxin A1 subunit, which covalently modifies R201 (Fig. 35.1). Transgenic expression of the cholera toxin A1 subunit in somatotrophs leads to pituitary hyperplasia and gigantism, whereas expression in thyroid cells leads to thyroid hyperplasia and hyperthyroidism.109 Gsα overexpression in the heart leads to cardiomyopathy,110 and expression of constitutively-activated forms of Gsα in the forebrain disrupts associative and spatial learning.111 A model of FD was created by transplanting Gsα-mutated skeletal progenitor cells into immunocompromised mice.112 A mouse model with germline expression of the R201C mutation survived, and with aging developed a skeletal dysplasia radiographically and histologically similar to FD.113View chapterPurchase book

Gsα, Pseudohypoparathyroidism, Fibrous Dysplasia, and McCune–Albright Syndrome

Lee S. Weinstein, … Allen M. Spiegel, in Genetics of Bone Biology and Skeletal Disease, 2013

Animal Models

Mouse models leading to constitutively activation of cAMP formation have been created by transgenic overexpression of Gsα, by expression of R201H or Q227L mutant forms of Gsα, or by expression of the cholera toxin A1 subunit which covalently modifies R201 (see Figure 27.1). Transgenic expression of the cholera toxin A1 subunit in somatotrophs leads to pituitary hyperplasia and gigantism, whereas expression in thyroid cells leads to thyroid hyperplasia and hyperthyroidism.73 Gsα overexpression in the heart leads to cardiomyopathy,74 and expression of constitutively-activated forms of Gsα in the forebrain disrupts associative and spatial learning.75 A model of FD was created by transplanting Gsα-mutated skeletal progenitor cells into immunocompromised mice.76 A mouse model replicating the full MAS phenotype has not been reported, perhaps because of the adverse effects of more generalized cAMP activation on normal development.View chapterPurchase book

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Evol Med Public Health. 2016; 2016(1): 170–176.Published online 2016 May 21. doi: 10.1093/emph/eow014PMCID: PMC4875775PMID: 27121451

Mouse models of human disease

An evolutionary perspectiveRobert L. Perlman*Author informationArticle notesCopyright and License informationDisclaimerThis article has been cited by other articles in PMC.Go to:

Abstract

The use of mice as model organisms to study human biology is predicated on the genetic and physiological similarities between the species. Nonetheless, mice and humans have evolved in and become adapted to different environments and so, despite their phylogenetic relatedness, they have become very different organisms. Mice often respond to experimental interventions in ways that differ strikingly from humans. Mice are invaluable for studying biological processes that have been conserved during the evolution of the rodent and primate lineages and for investigating the developmental mechanisms by which the conserved mammalian genome gives rise to a variety of different species. Mice are less reliable as models of human disease, however, because the networks linking genes to disease are likely to differ between the two species. The use of mice in biomedical research needs to take account of the evolved differences as well as the similarities between mice and humans.Keywords: allometry, cancer, gene networks, life history, model organisms

If you have cancer and you are a mouse, we can take good care of you. Judah Folkman [1]

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INTRODUCTION

Because of their phylogenetic relatedness and physiological similarity to humans, the ease of maintaining and breeding them in the laboratory, and the availability of many inbred strains, house mice, Mus musculus, have long served as models of human biology and disease [2]. Genomic studies have highlighted the striking genetic homologies between the two species [34]. These studies, together with the development of methods for the creation of transgenic, knockout, and knockin mice, have provided added impetus and powerful tools for mouse research, and have led to a dramatic increase in the use of mice as model organisms. Studies on mice have contributed immeasurably to our understanding of human biology [5]. All too often, however, mice respond to experimental interventions in ways that differ markedly from humans. Endostatin, the anticancer drug alluded to in the epigraph, is but one of many treatments that cure cancer in mice but have limited effectiveness in humans [6]. Indeed, the majority of oncology drugs that enter clinical trials never reach the marketplace. There are many reasons for the high failure rate of drug development, but the limitations of the animal models used in drug testing are an important factor [7]. Many substances that are carcinogens in mice are not carcinogenic in humans—and vice versa [8]. Moreover, mouse strains that were created to mimic human genetic diseases frequently have phenotypes that differ from their human counterparts [9]. Because of the assumption that mice will serve as reliable models for humans, differences between the two species are often greeted with surprise as well as dismay. But these differences should not elicit surprise; indeed, they should be expected. The lineages leading to modern rodents and primates are thought to have diverged from a common ancestral species that lived some 85 million years ago [10]. Since that time, species in these lineages evolved in and became adapted to very different environments. Our evolved developmental processes produce different kinds of organisms from similar component parts. Differences between mice and humans may be due to selection or drift, acting over the eons of evolutionary time or more recently during the creation and breeding of laboratory mouse strains [11].Go to:

SIZE

The most obvious and perhaps the most fundamental difference between mice and humans is size: humans are roughly 2500 times larger than mice. Size influences many aspects of an organism’s interactions with its environment, including its ability to acquire food, to avoid predators and to attract mating partners, and so has important effects on fitness; in the words of J. B. S. Haldane, organisms must be “the right size” [12]. As the lineages leading to mice and humans evolved, there was presumably selection for organisms that were the right size for their environments. Given its importance, size itself was probably a major target of natural selection [1314]. But a host of traits are correlated with size, and during the course of rodent and primate evolution, these traits evolved together with size. Two prominent sets of traits that are correlated with size are metabolic rate and life history strategy [15].Go to:

METABOLIC RATE

Metabolic rates of placental mammals are closely correlated with size. The relationship between basal metabolic rate (in kcal/day) and body mass (in kg) is usually taken as BMR = 70 × Mass0.75 [16]. Thus, a 30-g mouse has a specific metabolic rate (metabolic rate per gram of tissue) roughly seven times that of a 70-kg human [15]. There is continuing controversy about the reasons for the relationship between size and metabolic rate, and about the value of the allometric exponent [17]. The increased specific metabolic rate of small mammals is presumably related, at least in part, to size-dependent differences in heat loss and in requirements for thermoregulation, and is characterized by increases both in nutrient supply (capillary density) [18] and in nutrient demand (mitochondrial density) [19] in tissues of small animals; since nutrient supply and demand have coevolved and develop together during ontogeny, they are closely matched [20]. Differences in metabolic rate between mice and humans are correlated with many anatomic, physiologic and biochemical differences. Mice have relatively higher amounts of metabolically active tissues, such as liver and kidney, and relatively less inactive tissue, such as bone; in addition, mice have larger deposits of brown fat, which plays a critical role in heat production and thermoregulation. Mouse cells differ from human cells not only in mitochondrial density and metabolic rate, but also in the fatty acid composition of their membrane phospholipids; specifically, membranes in mouse cells have a higher content of the polyunsaturated (and readily oxidizable) fatty acid docosahexaenoic acid [21]. Mice have higher rates of production of reactive oxygen species and suffer higher rates of oxidative damage than do humans. All of these differences presumably evolved in association with selection for differences in size or in association with some other trait that is correlated with body mass, such as life history and rate of aging.Go to:

LIFE HISTORY

Size is also associated with a suite of life history traits, including age at reproductive maturity, length of gestation, litter size, birth interval, fraction of energy devoted to reproduction, and, perhaps most importantly, life expectancy. Female wild mice reach sexual maturity in a matter of 6–8 weeks, have a gestation length of 19–20 days and a litter size of 5–8, and produce multiple litters a year. Many laboratory mouse strains have been selected for increased fertility; they reach sexual maturity earlier and produce larger litters than do wild mice [22]. Mice, like other rodents, invest a much larger proportion of their energy in reproduction than do humans [23]. Both wild and laboratory mice have life spans of about 3–4 years, but wild mice have a much shorter life expectancy (less than a year, depending of course on environmental conditions) than do laboratory strains, which typically live several years [22]. Again, the differences in life history strategies between humans and mice are correlated with, and are probably related to, differences in size.Go to:

DIETS, MICROBIOMES AND PATHOGENS

Evolved differences in murine and human diets are also associated with pervasive differences in the biology of the two species. Although both mice and humans are omnivores, wild mice seem preferentially to consume unprocessed grains and cereals. Mice have large and continuously growing incisors that enable them to eat these foods. Presumably because their ancestors’ diets were low in ascorbic acid, mice have retained the ability to synthesize this essential cofactor; humans, in contrast, have lost this ability and so we now require exogenous vitamin C. And presumably because of their ancestors’ ingestion of different xenobiotics, mice and humans have different complements of cytochrome P450 enzymes and different patterns of xenobiotic metabolism [2425]. At least in part for this reason, toxicology testing in mice has been a poor predictor of human toxicity [26]. More importantly, mice have different microbiomes [27] and have coevolved with different sets of pathogens than have humans. The anatomy of the gastrointestinal track differs between the two species [27]. The ratio of length of the small intestine to that of the colon is greater in mice than in humans, mice have a prominent cecum, and they lack an appendix. In mice, the cecum is an important site for the microbial fermentation of undigested foods. Thus, the two species provide different environments that apparently support the growth of different gastrointestinal microbiota. Moreover, mice have significant amounts of bronchus-associated lymphoid tissue, which has been interpreted to indicate that, because they live close to the ground, they face increased exposure to respiratory pathogens in droplets or particles from the soil [28]. The differences between mice and humans are not only genetic and epigenetic, but also reflect features of their environments, especially their ecological interactions with other species (food sources, microbiota, pathogens, etc.) that are reliably transmitted from generation to generation and affect the course of development.Go to:

DIFFERENCES DUE TO THE DOMESTICATION AND BREEDING OF HOUSE MICE

During the course of murine and human evolution, our ancestors underwent selection for—and so mice and humans now differ in—many other traits, including circadian rhythm (wild mice are nocturnal), sensory systems (mice rely heavily on olfaction, hearing and touch), cognitive development, reproductive behavior and patterns of social organization. Moreover, the domestication and breeding of the laboratory mouse strains that are commonly used in biomedical research have increased the differences between the biology of these strains and that of wild mice, let alone human biology. Many laboratory mouse strains were derived from fancy mice, which had been kept as pets for centuries. These strains were derived largely from the subspecies M. musculus domesticus, which, for unknown reasons, has an exceptionally high rate of robertsonian chromosomal translocations [29]. Initially, domestication entailed selection for such traits as docility and the ability to thrive and reproduce in confinement. Later, as mouse breeding became a commercial enterprise, breeders selected for traits associated with increased reproduction, including early sexual maturity and the production of frequent and large litters [30].

A major impetus for the development of inbred mouse strains was to study the genetic basis of cancer; strains were created that differed in their susceptibility to transplanted tumors or in the incidence of spontaneous neoplasms [31]. These inbred strains have yielded many important insights into cancer biology. Nonetheless, cancer and other diseases in laboratory mice that were selected because they develop (or are resistant to) these diseases may differ from the cognate diseases found in wild mice, as well as from diseases in humans. Common strains of laboratory mice have come to differ from wild mice in a host of traits. Some of these differences, such as increased fertility, can be understood as the result of selection, while the reasons for other differences are not clear [30]. Finally, the genetic homogeneity that makes these strains valuable in the laboratory means, of course, that they lack the genetic variation that characterizes outbred wild populations.

Given the many differences in the biology of mice and humans, it is not surprising that the patterns of disease differ in the two species. The causes of death of feral house mice depend on the environment. Many are killed by predators, and in harsh environments starvation and hypothermia are major causes of death [22]. In the laboratory, mice live longer; there, cancer is a major cause of death, while cardiovascular disease is negligible. The distribution of tumors differs between mice and humans; most murine tumors are of mesenchymal origin, while human tumors arise mainly from epithelial cells. There are many other differences between mouse and human cancers, and many differences between mouse and human cells that appear to contribute to these differences [832–34]. For example, laboratory mouse strains have much longer telomeres than do humans and express telomerase in their somatic cells throughout life. This difference may help to explain why, in vitro, mouse cells undergo spontaneous transformation at much higher rates than do human cells.

Some of the differences between mice and humans are relatively easy to rationalize. As discussed below, differences in the function of the immune system have almost certainly evolved in response to differences in pathogen exposure and in life expectancy [28]. Other differences, such as differences in genomic imprinting, are harder to understand [35]. Additional phylogenetic analyses and functional genomic studies will be necessary to determine which of the differences between mouse and human biology are related to differences in size, either because they are associated with metabolic rate or with life history strategy, which are due to other changes that accompanied the evolutionary divergence of these species, and which have resulted from the selective breeding of laboratory mice.Go to:

IMPLICATIONS OF SPECIES DIFFERENCES FOR MOUSE RESEARCH

The use of model organisms in biological research is based on the concept of unity in biology, a concept expressed most famously in Jacques Monod and François Jacob’s aphorism, “Anything found to be true of E. coli must also be true of elephants” [36]. But biology is characterized by diversity as well as unity; evolution is “descent with modification” [37]. The art of choosing model organisms involves recognizing the properties of these organisms that they are likely to share with organisms of other species—especially, for biomedical research, humans [38]. Monod and Jacob were concerned with genetic regulatory mechanisms and other basic biological processes that must have arisen very early in the evolutionary history of living organisms and so are similar in bacteria and in mammals. Mice have served and will continue to serve as valuable models for the study of basic biological processes that, in Wimsatt’s terms, became developmentally entrenched before the rodent and primate lineages diverged and have been conserved during the separate evolutionary histories of mice and humans [39].

Studies of the immune system highlight both the value of mouse research in elucidating common features of mammalian biology as well as the limitations of translating this research in areas in which humans are likely to differ from mice. Research on mice has contributed greatly to our knowledge of the adaptive immune system; mouse research has led to the discovery of the major histocompatibility complex genes and the T cell receptor, and to our understanding of the regulation of antibody synthesis and many other features of the immune system [40]. But there are many differences between the mouse and human immune systems, such that much research on immunological diseases in mice is not transferable to humans, and many immunologists are now calling for a return to the study of human immunology [2840–42]. From an evolutionary perspective, this is understandable. The adaptive immune system evolved in jawed fish some hundreds of million years before the evolution of mammals. Many features of this ancestral immune system, including antigen recognition, generation of antibody diversity, clonal selection, and immunological tolerance, are critical for survival and have been maintained in most or all of the descendants of these early vertebrates. On the other hand, species differences in the mechanisms for the maintenance of memory T cells must have evolved in response to the evolution of different life spans. Moreover, specific features of the immune system evolve rapidly, as host species coevolve with their pathogens and commensal microbiota [41]. Since humans and mice harbor different sets of pathogens and microbiomes, it is not surprising that host–pathogen and host–microbiome coevolution has led to differences between the human and mouse immune systems.

The fact that the highly conserved mammalian genome can give rise to a wide variety of different species indicates that the relationships between genotype and phenotype differ among mammalian species. Comparisons between mice and humans are invaluable for understanding the developmental mechanisms that lead to such different genotype–phenotype relationships. Some of the genetic differences between mice and humans are differences in coding sequences, which give rise to proteins with different properties. For example, mouse hemoglobin has a lower affinity for O2 than does human hemoglobin, which facilitates the dissociation of O2 from hemoglobin in peripheral tissues and helps to support the higher metabolic rate in mice. Perhaps more importantly, however, are differences in the genetic or epigenetic regulation of gene expression in these species. The expression of potassium channel genes in the heart exemplifies these differences. Mice have a heart rate of ∼600 beats/min, while humans have a resting heart rate of ∼70 beats/min. This difference in heart rate entails that the cardiac action potential be much shorter in mice than in humans. Indeed, the repolarization phase of the cardiac action potential, which is due to outward Kcurrents, is much shorter in mice [43]. This difference is due to different contributions of various Kcurrents, which in turn are presumably due to differences in expression of Kchannel genes in the two species. Evolved differences in the regulation of gene expression are important because they may involve the rewiring of gene (or protein) networks. Gene networks in mice and humans have similar numbers of nodes (genes) but the connectivity of the nodes in these networks, and the relationships between genes and phenotypes, differ between the two species [44–46]. The different network architectures and different genotype–phenotype relationships between mice and humans mean that the relationships between genotype and disease are also likely to differ in these two species. Perturbations of gene and protein networks by environmental manipulation as well as by mutation are likely to have different effects on diseases as well as on other phenotypes in mice than in humans. In short, mice are problematic models for understanding human disease.

There are other good reasons to pursue research on mice. Although house mice are not a major source of human disease, they can transmit lymphocytic choriomeningitis virus and perhaps other pathogens to humans, and other rodent species are important reservoirs for zoonoses. Research on mice may yield information that will help to prevent or ameliorate these diseases. Finally, mice should be studied for their own sake, to understand their biology and to maintain the health of pet mice, laboratory mice, and wild mice.

Unfortunately, despite the many attempts to translate the results of mouse research to humans, we still cannot specify in advance which research in mice is likely to benefit or shed light on human biology and health. For the most part, we have only anecdotal information about studies in mice that translated to humans and those that did not. We need more systematic collection, reporting and analysis of mouse research (and research on other “model organisms”) to figure out what works and what does not. Until we have that information, we need to be more critical in pursuing mouse research and in making claims about the applicability of this research to humans.

In addition to problems resulting from the evolved differences between mice and humans, other aspects of mouse research have compromised the value of this research and have further complicated the extrapolation of mouse research to humans. Thus, e.g., laboratory mice are often housed at temperatures below their thermoneutral zone, and as a result are cold-stressed, sleep deprived, and hypertensive [47]. The biology of laboratory mice may also be affected by their housing in same-sex groups and their lack of opportunities for physical exercise. Although mice are often used as models of diseases of aging, for logistical and financial reasons most mouse research is carried out on young animals. And although mouse cells are more sensitive to oxygen damage than are human cells, cell culture studies are often carried out in 20% oxygen, which is non-physiological and is more damaging to mouse cells than to human cells [48]. Finally, there are no agreed upon standards for the design, analysis, or publication of mouse research (or research with other model organisms). The statistical analysis of studies of mice and other animals is often substandard, and there may be important publication biases because negative results may not get published [4950]. All of these problems need to be addressed before studies on mice can be properly interpreted and extrapolated to humans.Go to:

FINAL COMMENTS

Despite all of the documented differences between mice and humans, and despite the history of “errors in translation” in the application of research on mice to humans, reports of research on mice are frequently accompanied by unwarranted and misleading claims, such as “Furthering our understanding of mouse X should provide novel insights into human Y.” Such claims raise false hopes and are ultimately self-defeating, in that they waste resources and increase public skepticism concerning the value of biomedical research. Indeed, the problems of translating research on mice and other model organisms to humans have led a number of scientists to question the value of this research [51–53]. Furthermore, critical discussions of animal experimentation are routinely distorted by “animal rights” activists to support their belief that this experimentation should be stopped. These intrusions, however unwelcome, should not stifle discussion. For reasons mentioned above, research on mice (and other species) is essential and should be supported. This research should, however, be designed and interpreted with appropriate appreciation of the evolved differences as well as the similarities between M. musculus and H. sapiens.Go to:

ACKNOWLEDGEMENTS

I thank Alan Schechter and Ted Steck for their thoughtful comments and helpful suggestions.

Conflict of interest: None declared.Go to:

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J Biomed Biotechnol. 2011;2011:497841. doi: 10.1155/2011/497841. Epub 2011 Feb 16.

Animal models of cardiovascular diseases.

Zaragoza C1Gomez-Guerrero CMartin-Ventura JLBlanco-Colio LLavin BMallavia BTarin CMas SOrtiz AEgido J.

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1Department of Epidemiology, Atherothrombosis and Cardiovascular Imaging, Fundacion Centro Nacional Investigaciones Cardiovasculares Carlos III (CNIC), Sinesio Delgado 3, 28029 Madrid, Spain.

Abstract

Cardiovascular diseases are the first leading cause of death and morbidity in developed countries. The use of animal models have contributed to increase our knowledge, providing new approaches focused to improve the diagnostic and the treatment of these pathologies. Several models have been developed to address cardiovascular complications, including atherothrombotic and cardiac diseases, and the same pathology have been successfully recreated in different species, including small and big animal models of disease. However, genetic and environmental factors play a significant role in cardiovascular pathophysiology, making difficult to match a particular disease, with a single experimental model. Therefore, no exclusive method perfectly recreates the human complication, and depending on the model, additional considerations of cost, infrastructure, and the requirement for specialized personnel, should also have in mind. Considering all these facts, and depending on the budgets available, models should be selected that best reproduce the disease being investigated. Here we will describe models of atherothrombotic diseases, including expanding and occlusive animal models, as well as models of heart failure. Given the wide range of models available, today it is possible to devise the best strategy, which may help us to find more efficient and reliable solutions against human cardiovascular diseases.PMID: 21403831 PMCID: PMC3042667 DOI: 10.1155/2011/497841[Indexed for MEDLINE] Free PMC Article

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Am J Cardiovasc Dis. 2016; 6(3): 70–80.Published online 2016 Sep 15.PMCID: PMC5030387PMID: 27679742

Small mammalian animal models of heart disease

Paula Camacho,1Huimin Fan,2Zhongmin Liu,2 and Jia-Qiang He1Author informationArticle notesCopyright and License informationDisclaimerThis article has been cited by other articles in PMC.Go to:

Abstract

There is an urgent clinical need to develop new therapeutic approaches for treating cardiovascular disease, but the biology of cardiovascular regeneration is complex. Model systems are required to advance our understanding of the pathogenesis, progression, and mechanisms underlying cardiovascular disease as well as to test therapeutic approaches to regenerate tissue and restore cardiac function following injury. An ideal model system should be inexpensive, easily manipulated, reproducible, physiologically representative of human disease, and ethically sound. The choice of animal model needs to be considered carefully since it affects experimental outcomes and whether findings of the study can be reasonably translated to humans. This review presents a guideline for the commonly used small animal models (mice, rats, rabbits, and cats) used in cardiac research as an effort to standardize the most relevant procedures and obtain translatable and reproducible results.Keywords: Mouse, rat, rabbit, cat, myocardial infarction, heart failure, diabetic cardiomyopathyGo to:

Introduction

Cardiovascular disease is considered the major cause of morbidity and mortality worldwide. Over the past several years, enormous achievements have been made in the management of cardiovascular disease, which has depended on the use of experimental animal models [1]. However, there are still no permanent cures for most cardiovascular diseases. For example at present, the only cure for heart failure (HF) is a heart transplant; however, its therapeutic potential is limited by very small numbers of donor hearts available relative to the need and is complicated by long-term allograft vasculopathy. Tissue-engineered biomaterials and autologous or allogeneic cell transplant therapy may have the potential to restore function and obviate the necessity for transplant [1,2]). In recent years, gene therapy and cell transplant technologies have advanced remarkably. If proven efficient and safe, these therapies might revolutionize the treatment of myocardial failure [3].

A major challenge of preclinical testing is to establish clinically relevant models for myocardial failure and infarction [3]. Donated human hearts, either non-transplantable (ranging from healthy to diseased) or end-stage failing hearts, typically obtained at the time of transplantation, are great tools for addressing this issue. These tissues are, however, available in very limited quantities and exhibit great variability due to differences in factors such as genetics, medication, diet, social habits, and disease. Therefore, it is necessary to have a suitable animal model where cardiac physiology and disease can be studied efficiently and reliably with translational applicability to humans [4].

Depending on the cardiovascular process being studied, the choice of animal model needs to be considered carefully since it affects experimental outcomes and whether findings of the study can be reasonably translated to humans. As a simple rule, the closer the heart or body weight of the animal model to human heart or body weight, the more similar are the hearts [4]. Both small and large animal models of heart disease have advantages and disadvantages. The main problems with using small animals are how to establish functional benefit of the treatment and the translation of results. In large-animal models, the ethical requirement for limiting the number of animals influences study protocols, especially concerning non-human primates or dogs [3].

Determining the best experimental model of a human condition requires a number of decisions and compromises – especially in relation to obtaining the optimal balance between the quantity and quality of the data produced and the relevance of the data to the condition under investigation. In assessing the utility of an investigative model, it is necessary to identify the research objectives. The nature of the question under study will greatly influence selection of the most appropriate investigative models and endpoints of cardiac function and malfunction [5]. It is also important to standardize the procedures used so as to obtain relevant and reproducible results that can be compared with other findings.

Rodent models are often used in cardiovascular research since they are easier to handle and house, have a short gestation time, genetically manipulate to generate transgenic strains, and have low maintenance costs; therefore, they are more suitable for “high-throughput” studies than large animal models [6]. These characteristics make small rodent models the most used model for cardiac physiology and disease, genetics, pharmacology, and long-term survival studies [4]. However, rodents are phylogenetically very distant from humans and some pathophysiological features of disease and their response to pharmacological treatments may not be reliable predictors [4,6].Go to:

Mouse

During the past 15 years, the mouse has become the model organism of choice to study human heart disease. 99% of human genes have direct murine orthologs and mice are suitable for selection of genetically modified individuals within a relatively short time because of their high breeding rate. They also have a short life span, allowing investigators to follow the natural history of the disease at an accelerated pace [6]. Use of genetically modified mouse models allows for rapid establishment of proof-of-principle, which can later be extended into larger animal models and, eventually, into humans [4].

Despite their widespread use, mice represent a heart model that is farthest from human contractile function, mainly due to their small size and short lifespan. Thus, translational aspects and the value of genetic mouse models must be interpreted with caution. Although these models may recapitulate some of the characteristics of the human cardiac phenotype of a disease, they typically do not recapitulate all aspects of human cardiovascular disease [4].

Ligation-induced myocardial infarction (MI)

MI animal model is one of the most commonly used one to mimic human heart attack. Mortality after MI is relatively high and surviving patients are often severely compromised due to insufficient heart-pump function. At the cellular level, damage to the heart’s contractile constituents, the cardiomyocytes, is irreversible, and treatments merely serve to reduce symptoms. Only a minority of patients receives heart transplants [7].

The neonatal mouse MI model represents a useful tool for evaluating mammalian cardiac regeneration and cellular/molecular mechanisms that govern cardiomyocyte proliferative capacity only in the early stage of neonatal mice [8]. To generate MI, neonates are briefly sedated using an isoflurane induction chamber followed by a short cooling period (a few minutes) in ice water. The animals are then taped to a cooling bag in the right lateral position to ensure continued hypothermic anesthesia during the surgical procedure. The skin is cut a few millimeters below the left foreleg, the thorax opened in the 4th intercostal space, and the left anterior descending (LAD) artery is ligated. MI is indicated by a light pallor of the myocardium below the ligature after suturing. The musculoskeletal thorax and the skin are closed, and mice are put onto a 37°C heated pad to recover from the anesthesia [810].

It is commonly found that the complete regeneration of cardiomyocytes can be reached when the LAD artery is ligated in newborn mice. Myocardial necrosis is usually observed at Day 3 after ligation as ~75% of the myocardium below the ligature becomes nonviable. There is also a marked decline in left ventricular (LV) systolic function at Day 4 after injury. Cardiac systolic function is able to restore to sham operating levels at Day 21 after MI as the myocardium is repaired to greater than 95% viable [8]. However, the complete regeneration is age-dependent. For 1 day old mice, they are able to clear fibrotic tissue and replenish all cardiomyocytes while for 7 days old mice, their capacity for the capacity for complete morphological and functional cardiac regeneration was lost and retained significant scar tissue at day 21 after MI [9]. Very few proliferating cardiomyocytes were identified in 7 day old mice after MI and the remodeling response of 14 day old mice was characterized by extensive fibrosis, wall thinning, and ventricular dilation after MI [8].

The adult mouse infarct model better represents the cardiac response of human patients to an ischemic injury than neonatal mouse model. To induce heart infarction, mice are anesthetized by inhaling isoflurane or injecting anesthetics (e.g., Ketamine/Xylazine). The mice are then intubated using a catheter and ventilated with a rodent ventilator. The surgical procedure is similar to the one described above for neonatal mice. A left-sided thoracotomy is performed and the LAD artery is permanently ligated, approximately ~4 mm distal to the origin of the artery under the left atrium. Infarction is verified by LV blanching distal to the suture. The chest and skin are then closed, and mice are allowed to recover in a heated chamber [8,11,12]. Inflammation and scar tissue formation replacing the necrotic myocardium after MI are essential to preventing cardiac wall rupture. It is possible that ischemic injury, with subsequent cell necrosis, apoptosis, and damage to the extracellular matrix, dictates the course of inflammation and inhibits complete regeneration [13].

An inherent limitation of the LAD ligation model both in neonates and adults makes standardizing injury size difficult. Given that a proximal LAD occlusion can result in infarction of as much as 40% of the LV, it is highly plausible that larger ischemic injuries, similar to larger resection injury, are incompatible with complete regeneration [14].

Cryogenic injury

Cryogenic injury is another mouse model for studying heart regeneration and cellular remodeling. Unlike MI, the cryoinjury model induces confluent necrosis but does not spare cells within the center of the lesion. It can also have interdigitating viable and necrotic tissue [15]. Interestingly, cardiomyocytes are capable to proliferate sufficiently to effectuate myocardial regeneration after extensive cryoinjury of the LV myocardium in adult mice. High levels of macrophages precede and coincide with the highest levels of cardiomyocyte proliferation, suggesting a functional role of these cells in myocardial regeneration [16].

Cryogenic injury can be induced in several places throughout the heart, depending on the investigators’ area of interest. An open thoracotomy approach is used to induce cryoinjury on the LV, because this allows direct visualization of the freezing process and immediate validation of the resulting freeze-thaw injury. A liquid nitrogen-cooled aluminum probe is applied directly onto the anterior LV for ~10 seconds. The frozen myocardium thaws completely 10-15 seconds following probe removal, and the injured area exhibits a deep red coloration [12]. Three 1-min exposures cause an extensive lesion on the midventricular portion of the anteriolateral LV wall [11].

Another approach is through an abdominal incision to induce cryoinjury on the right ventricle (RV). Mice are anesthetized and the abdomen is opened through a transverse laparotomy. The translucent diaphragm is exposed by lifting the chest with a divaricator. The surface of the heart is now visible through the diaphragm and the desired area for the injury can be selected. A blunt tip stainless steel Von Graefe hook is directly placed on the diaphragm after cooling for ~2 min in liquid nitrogen to make an apical or midventricular RV injury. Two sequential 10-s exposures (with an intermittent 30-s interval) cause an extensive, transmural lesion on the lateroapical portion of the RV wall with an extension to the apical LV wall. A single 30-s exposure causes an extensive, transmural lesion on the midventricular RV wall with no extension to the septum [11]. This procedure has also been described in rats [17].

Doxorubicin (DOX)-induced HF

Nonischemic cardiomyopathy accounts for approximately one-third of HF cases. Unlike ischemic forms, which are amenable to palliative procedures like revascularization and remodeling operations, nonischemic cardiomyopathies, once they have reached an end stage of drug refractoriness, can only be treated radically by heart transplantation. DOX-treated mice receive a single dose of DOX at 15-20 mg/kg i.p., which causes clinical symptoms of HF [18]. Mice are studied five days later because at this time point more than 5 final half-lives of elimination of DOX from both plasma and cardiac tissue in mice has taken place and, at the time of functional and immunohistochemical assessment, DOX is no longer present in the blood or cardiac tissues. LV fractional shortening and cardiac output are significantly reduced (35 and 23%, respectively) relative to control animals. This murine model of severe LV dysfunction by DOX cardiotoxicity mimics the human pathology. Changes are consistent with clinical observations, suggesting that this murine model is appropriate for mechanistic evaluations [19].

Transgenic lines

One of the most advantageous aspects of utilizing mice is the ability to make genetic models. Although such models can be produced in larger species, mouse models can be developed in a shorter time period due to their short gestation age at a substantially lower cost [4].

Dilated cardiomyopathy (DCM)

Two strains of mice have been established to model the development, progression, and regression of DCM in humans. Muscle LIM protein (MLP) null mice are engineered by a deletion of MLP, an actin-associated cytoskeletal protein and Casequestrin (CQS) mice have a cardiac-restricted overexpression of the calcium binding protein CQS. Both produce a HF phenotype that replicates various aspects of human DCM. The MLP-/- mice appear to closely replicate human DCM, with progressive reduction in cardiac function beginning at 4-6 months, whereas CSQ mice develop rapid-onset DCM resulting in mortality by 9-14 weeks [20]. Echocardiography shows normal contractility in 4-week-old MLP null mice and a progressive decrease in contractility with increasing age. This suggests that contractile dysfunction is not the primary defect in this mouse model of DCM. Rather, the lack of MLP causes hypertrophy, resulting in HF with an increase in connecting tissue and increased myocardial stiffness [21].

Hypertrophic cardiomyopathy (HCM)

Mouse models in which cMyBP-C is homozygously ablated (cMyBP-C/) have been used to study the role of cMyBP-C in normal cardiac contractility and the development of HCM. Alterations in contractile kinetics serve as the primary pathophysiologic trigger for the development of hypertrophy in this model [22]. Another transgenic mouse model of HCM that overexpresses myotrophin in the heart, the Tg mouse, was also developed. This model showed hypertrophy as early as 4 weeks of age that progressively led to HF with severe compromised function. All the symptoms in this model mimic human HF. Tg mice demonstrated prevalent DNA damage in hearts during transition from long standing hypertrophy to HF and the induction of the cell regenerative machinery, especially in cardiac myocytes. Human HCM heart samples show similar changes in several genes for apoptosis and cell regeneration. Data in the Tg mouse model convincingly suggest that cell death and regeneration take place simultaneously during the transition of hypertrophy to HF [23].

Autoimmune cardiomyopathy (AICM)

To develop an animal model for AICM, DQ8 transgenic non obese diabetic (NOD) mice were crossed with a NOD Major Histochmpatibility Complex (MHC) class II β-chain knockout (KO) line. Animals from the original DQ8 transgenic NOD line developed spontaneous autoimmune diabetes at the same rate as the regular NOD/LtJ strain. However, when the DQ8-NOD animals were crossed with the NOD MHC class II KO line, the resulting DQ8+/+, IAβ/ NOD animals no longer developed diabetes or insulitis; instead, they developed progressive DCM (hearts three to four times than normal size) and HF leading to premature death in both males and females. As the animals aged, first-degree atrioventricular (AV) block became widespread in the population, and by 18 weeks, the majority of animals had progressed to second degree or complete AV block. Histological examination of end-stage hearts showed pancarditis, with grossly dilated atria and only a few live cardiomyocytes remaining in mostly fibrotic atrial walls, which in places were paper thin [24].

Duchenne muscle dystrophy (DMD)

DMD is an X-linked neuromuscular disorder caused by a mutation in the dystrophin gene [25]. Although limb muscle weakness and loss of ambulation are usually the initial clinical signs of the disease, patients with DMD die from respiratory failure or HF. To improve lifespan and quality of life, progressive loss of contractile function in the heart also needs to be prevented or halted [26].

The genotypic murine model for DMD is the dystrophin-deficient Mdx mouse. The disease phenotype is milder in Mdx mice in contrast to human counterparts [25]. They have a normal life span and do not show significant muscle weakness or cardiac alterations nor any of the skeletal or cardiac muscle lesions that develop at late time points in DMD patients, such as fibrosis and muscular atrophy [27]. This is partially due to the compensatory effects of utrophin, a dystrophin homolog that can offset the loss of dystrophin in mice [26]. Another transgenic mouse was developed to better model the severe cardiac dysfunction in humans with DMD. In this double knock-out mouse, where both dystrophin and utrophin are absent, cardiac contractile function is severely affected at 8 weeks-of-age, displaying the classic pathophysiological hallmarks of end-stage human cardiac failure [28].

Several studies using dystrophin-deficient mice bred with other strains were conducted to clarify mechanisms leading to cardiomyopathy in DMD. Different therapies have been used to treat the symptoms of cardiomyopathy in DMD, but these agents cannot replace lost or damaged cardiomyocytes resulting from lack of fully functional dystrophin. Generation of new cardiomyocytes in dystrophin-deficient cardiac muscle from an endogenous population of cardiac stem cells suggests a potential mechanism that may be exploited to delay or prevent DCM in DMD [29]. Macrophages are necessary for repair of damage in the heart, as they stimulate new cardiomyocytes [29]. Also, IL-10 might be an important immune-modulator in dystrophic muscle. Cardiac inflammation induced by IL-10 ablation induced cardiac dysfunction and decreased LV function with LV and RV dilatation [30].

Atrial fibrillation (AF)

Cardiac-specific LKB1 KO mice are used to study AF and associated pathologies. 95% of KO mice developed AF by 12 weeks. They also demonstrated variable heart rhythm disorders including first- and second-degree AV block, bundle branch block, premature atrial and ventricular contractions, and atrial flutter. AF resulted in increased fibrosis, apoptosis, and disrupted ultrastructure [31]. KO mouse heart developed dilated atrial cardiomyopathy, which was vulnerable to electrical and structural remodeling. Atrial dilation, stretch, fibrosis, loss of muscle mass, cellular and matrix remodeling, and disruption of gap junctions are documented in this model. AF caused LV systolic dysfunction with depressed LV ejection fraction and clinical HF. It accurately represents human AF with characteristic structural and electrical remodeling in atria [31].Go to:

Rat

Ligation-induced myocardial infarction (MI)

The procedure to induce rat MI is very similar to the one described above for mice. Briefly, after rats are anaesthetized, intubated, and connected to a ventilator, a left thoracotomy is performed in the fourth intercostal space, the heart exposed, and the LAD artery ligated 2-3 mm from its origin between the pulmonary artery conus and the LA [3234].

Overload-induced cardiac hypertrophy

One of the most common and successful surgical models to create pressure overload HF is constriction of the ascending aorta. This model is clinically relevant because of its slow but steady progression from compensated cardiac hypertrophy to the decompensated phase and, finally, to the HF stage. Ascending aortic constriction can be performed in two ways, either by using sutures or by application of metallic clips, which is less surgically complicated. In this procedure, one has to locate the supra-valvular ascending aorta and insert the clip around the aorta to obtain the desired level of constriction [35,36].

Diabetic cardiomyopathy (DbCM)

Little attention has been devoted to defining the initial response of myocardial tissue to a short period of hyperglycemia in terms of proliferative properties of myocytes and alterations of cardiac stem cell storage before the appearance of the cardiomyopathic phenotype. A detailed knowledge of changes occurring at the very beginning of diabetes, when cardiac electromechanical performance is still normal, may suggest appropriate therapeutic approaches aimed at preventing the development of mechanical dysfunction and arrhythmogenesis, which characterizes more advanced stages of DCM [37].

To create an animal model for diabetes, a single intra-peritoneal injection of streptozotocin (STZ, 60 mg/kg) is applied to rats. This is one of the most commonly used experimental models of diabetes. Rats exhibit hyperglycemia and hyperlipidemia coupled with hyperinsulinemia [38]. Ventricular dysfunction and marked structural damage appear 12 weeks after STZ treatment. The first detrimental effect of metabolic changes is a marked loss of ventricular mass, in the absence of cardiomyocyte hypertrophy or accumulation of extracellular matrix [37]. However in STZ-injected rats, it is difficult to definitively exclude the possibility that the pathogenesis is related, at least in part, to a permanent toxic action of STZ [39].

Transgenic lines

MI in hypertensive rats

Coronary occlusion in spontaneously hypertensive rats (SHR) presents many similarities to MI in humans and represents an adequate model for the investigation of the role and mechanisms of stem cell therapy [40]. Hypertension in SHR causes higher LV systolic pressure, blood pressure, and RV and LV weight compared with their normotensive counterparts. After MI, hypertension accelerates LV dilatation and haemodynamic alterations, indicating that this animal model presents many similarities to MI in humans [32,40].

Type II diabetic rats

A rat model of Type II diabetes is the Goto-Kakizaki (GK) rat. GK rats have a stable, inheritable form of Type II diabetes characterized by mild hyperglycemia and hyperinsulinemia, with no obesity, hypertension, or marked hyperlipidemia. Under normoxic conditions, cardiac function in the GK rat is indistinguishable from that of control rats. A contractile defect in both systolic and diastolic LV function can be elicited in the GK heart during even brief moderate hypoxia. Regardless the level of insulin, a high level of extracellular glucose is sufficient for development of contractile abnormalities in diabetic cardiomyocytes, which may explain the absence of these abnormalities in the GK rat model where the level of hyperglycaemia is mild. Contractile dysfunction in the GK heart has a primarily metabolic basis [39].

Another rat model is the JCR: LA-cp rat. It is a normotensive, hyperinsulinaemic model of Type II diabetes, which differs from the GK rat in being obese and markedly hyperlipidaemic. In addition, these rats develop early atherosclerotic lesions of major blood vessels and occlusive coronary thrombi at later stages of the disease [39].

Duchenne muscular dystrophy (DMD)

A new model of dystrophin-deficient rats was made by microinjecting a mixture of TALE nuclease mRNA for DMD in rat zygotes, allowing for the generation of two DMD/Mdx rat lines. The lesions in heart and skeletal muscle in this model closely mimics those observed in DMD patients. As observed in DMD patients but not in Mdx mice, fibrosis is severe in all skeletal muscle examined as well as in cardiac muscle of DMD/Mdx rats. Cardiac muscles are affected with necrosis and fibrosis and show signs of progressive DCM. Echocardiography showed significant concentric remodeling and alteration of diastolic function. These results indicate that DMD/Mdx rats represent a new, invaluable small animal model for pre-clinical research on DMD [27].Go to:

Rabbit

Spontaneous watanabe heritable hyperlipidemic myocardial infarction (WHHL-MI)

Rabbit myocardium shares more similarities with human myocardium than small rodent myocardium; therefore, rabbit genetic models, albeit expensive, can be used as a steppingstone to determine whether a particular study can be extended to humans and larger animal models. Although, other large species such as canine and sheep more closely resemble the human heart, the cost of acquiring and housing rabbits is still significantly lower, making them an attractive alternative to larger animal models for cardiac research. Differences between rabbit and human myocardium remain, possibly resulting in a particular study or therapeutic intervention having differential effects in rabbits and humans. For example, rabbits might not serve as the best animal model for studying the effects of exercise on the cardiovascular system mainly because its heart rate reserve is much less than that in human and large animal models, such as canines, which would serve as better animal models in such cases [4].

A rabbit spontaneous MI model could be obtained from the selective breeding descendants of coronary atherosclerosis-prone WHHL-MI rabbits. Because the coronary lumen area stenosis was enhanced in these rabbits, the incidence of spontaneous WHHL-MI was increased in proportion to the serial nearly-occluded coronary lesions. Coronary plaques with complicated lesions are the major determinant for MI in these rabbits. However, observations indicate that the mechanisms for MI in WHHL rabbits are different from those in humans [41].Go to:

Cats

Hypertrophic cardiomyopathy (HCM)

Spontaneous cardiac diseases similar or identical to those in humans are extremely common in companion animals and are vastly underutilized as models of human cardiac disease. HCM is currently the most common heart disease in cats, and its incidence appears to be increasing. As in humans, feline HCM is characterized by hypertrophy of the (LV, impaired diastolic filling, secondary left atrial enlargement is usually evident, and variable right heart enlargement or hypertrophy may develop over time. Arrhythmias are uncommon unless severe left atrial enlargement is present. In cats with HCM, congestive HF and arterial thromboembolism are common clinical manifestations [42].

A specific breed, the Maine Coon, has a genetic mutation that makes the breed prone to suffer from HCM. The prevalence of HCM in cats homozygous for this mutation is much higher than in cats heterozygous for the mutation. However, it is highly likely that other causes (genetic or not) are also responsible for the disease in the Maine Coon [43]. Individuals of this breed can be used as relevant animal models to study the development and pathophysiology of HCM.Go to:

Conclusions

Small animal models are commonly used in cardiovascular research because of their many advantages over large animal models (see Table 1). They have a short life span, allowing the investigators to follow the natural history of the disease at an accelerated pace. Also, the development of genetically modified models allows for rapid establishment of proof-of-principle that can later be extended into larger animal models. However, rodents have a few disadvantages as well. They are phylogenetically very distant from humans and some pathophysiological features of disease and their response to pharmacological treatments may not be reliable predictors for humans. Thus, translational aspects and value of genetic small animal models must be interpreted with caution. Although they may display some of the characteristics of human cardiac disease, they typically do not recapitulate all aspects of it.

Table 1

Advantages and disadvantages of small animal models

AdvantagesDisadvantages
• Lower maintenance costs• Phylogenetically distant from humans
• Easier to handle and house• Pathophysiology of disease may not be translatable to humans
• Shorter gestation time and lifespan• Different response to pharmaceutics
• Suitable for proof-of-concept and “high-throughput” studies• Not suitable for chronic studies
• ischemia-reperfusion induced arrhythmias are infrequent and easy to reverse when they occur 
• Suitable for genetic selection and production of transgenic strains 

Determining the best experimental model of a human condition requires a number of decisions and compromises – especially in relation to obtaining the optimal balance between the quantity and quality of the data produced vs the relevance of the data to the condition under investigation. In assessing the utility of an investigative model, it is necessary to identify the research objectives. The question under study will greatly influence the choice of the most appropriate model and determination of cardiac function and malfunction. It is also important to standardize the procedures used in order to obtain relevant and reproducible results that can be compared with other findings. Table 2 summarizes the most relevant small animal models for cardiac disease and whether the disease is artificially induced or spontaneous. This provides a basis in which to decide what model best suits a specific investigation.

Table 2

Relevant small animal models for cardiac disease (only one reference is listed for each model. See the Main text for more references)

AnimalsInduced modelsSpontaneous models
MouseCryoinjury [11]AF [31]
DOX induced HF [18]AICM [24]
MI [8]DCM [21]
 DMD [28]
 HCM [23]
RatCardiac hypertrophy [35] Cryoinjury [17]Hypertensive rat [40] DbCM [39]
DbCM [38]DMD [27]
MI [33] 
Rabbits WHHL-MI rabbits [41]
Cats HCM [42]

Abbreviations: AF: Atrial fibrillation, AICM: Autoimmune cardiomyopathy, DbCM: Diabetic cardiomyopathy, DCM: Dilated cardiomyopathy, DMD: Duchenne muscular dystrophy, DOX: Doxorubicin, HF: Heart failure, HCM: Hypertrophic cardiomyopathy, MI: Myocardial infarction, WHHL-MI: Watanabe heritable hyperlipidemic myocardial infarction.Go to:

Acknowledgements

We thank Dr. Janet Webster of the Fralin Life Science Institute at Virginia Tech for providing a review of the manuscript. This work was supported by the Institute for Critical Technology and Applied Science (ICTAS) at Virginia Tech #JFC2014_JIAHE958451025). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.Go to:

Disclosure of conflict of interest

None.Go to:

Abbreviations

AFatrial fibrillationAICMautoimmune cardiomyopathyAVatrioventricularCSQcalsequestrinDbCMdiabetic cardiomyopathyDCMdilated cardiomyopathyDMDDuchenne muscular dystrophyDOXdoxorubicinGKGoto-KakizakiHCMhypertrophic cardiomyopathyHFheart failureKOknockoutLADleft anterior descendingLVleft ventricleMHCmajor histocompatibility complexMImyocardial infarctionMLPmuscle LIM proteinNODnon obese diabeticRVrights ventricleSHRspontaneously hypertensive ratSTZstreptozotocinWHHL-MISpontaneous Watanabe heritable hyperlipidemic myocardial infarctionGo to:

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Journal of Biomedicine and Biotechnology
Volume 2011, Article ID 497841, 13 pages
http://dx.doi.org/10.1155/2011/497841

Review Article

Animal Models of Cardiovascular Diseases

Carlos Zaragoza,1Carmen Gomez-Guerrero,2Jose Luis Martin-Ventura,2Luis Blanco-Colio,2Begoña Lavin,1Beñat Mallavia,2Carlos Tarin,2Sebastian Mas,2Alberto Ortiz,2 and Jesus Egido2

1Department of Epidemiology, Atherothrombosis and Cardiovascular Imaging, Fundacion Centro Nacional Investigaciones Cardiovasculares Carlos III (CNIC), Sinesio Delgado 3, 28029 Madrid, Spain
2Renal and Vascular Research Laboratory, IIS-Fundacion Jimenez Diaz, Universidad Autonoma, Avda Reyes Catolicos 2, 28040 Madrid, Spain

Received 11 October 2010; Revised 4 January 2011; Accepted 17 January 2011

Academic Editor: Oreste Gualillo

Copyright © 2011 Carlos Zaragoza et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Cardiovascular diseases are the first leading cause of death and morbidity in developed countries. The use of animal models have contributed to increase our knowledge, providing new approaches focused to improve the diagnostic and the treatment of these pathologies. Several models have been developed to address cardiovascular complications, including atherothrombotic and cardiac diseases, and the same pathology have been successfully recreated in different species, including small and big animal models of disease. However, genetic and environmental factors play a significant role in cardiovascular pathophysiology, making difficult to match a particular disease, with a single experimental model. Therefore, no exclusive method perfectly recreates the human complication, and depending on the model, additional considerations of cost, infrastructure, and the requirement for specialized personnel, should also have in mind. Considering all these facts, and depending on the budgets available, models should be selected that best reproduce the disease being investigated. Here we will describe models of atherothrombotic diseases, including expanding and occlusive animal models, as well as models of heart failure. Given the wide range of models available, today it is possible to devise the best strategy, which may help us to find more efficient and reliable solutions against human cardiovascular diseases.

1. Introduction

Cardiovascular diseases are the first leading cause of death and morbidity in developed countries. Cardiac and vascular complications are complex multifactorial pathologies, in which both genetic and environmental factors are implicated, thus making them very difficult to prevent. The development of animal models of cardiovascular disease (CVD), including cardiac and atherothrombotic diseases, has provided us today with important insights into the pathophysiology, and they were found to be essential tools to evaluate new therapeutic strategies to predict and to prevent these complications.

Here, we will summarize the most common models of cardiovascular diseases, including those implemented in both large and small animals, designed for helping to cover with more precision and to better understand every single aspect related to these human pathologies. In particular, we will describe models of atherothrombotic diseases, including expanding abdominal aortic aneurysms (AAA), thoracic aneurysms, and occlusive atherosclerotic diseases, as well as models of heart failure. These situations constitute today a significant challenge since predictors to evaluate early detection and forecast progression are crucial in these pathologies, yet they are poorly explored.

2. Animal Models of Atherothrombotic Disease

2.1. Mouse Models

Atherosclerosis is a complex multifactorial disease with different etiologies that synergistically promote lesion development. Mouse models have proved to be useful to study development and progression of atherosclerotic lesion, and several reviews have extensively discussed the different available models [13]. In particular, knockout and transgenic mouse models for atherosclerosis have been instrumental in understanding the molecular and cellular mechanisms involved in atherogenesis, and in evaluating the effectiveness of new and existing atherosclerotic drugs [4].

As wild-type mice are resistant to lesion development, the current mouse models for atherosclerosis are based on genetic modifications of lipoprotein metabolism with additional dietary changes. Among them, low-density lipoprotein receptor-deficient mice (LDLR−/− mice) and apolipoprotein E-deficient mice (apoE−/− mice) are the most widely used. Atherosclerotic lesions seen in these models can be exacerbated by the addition of risk factors such as hypertension or diabetes. Mice have become widely used as models of human atherosclerosis as they offer advantage compared with other species (Table 1).

tab1

Table 1: Animal models of atherosclerosis: advantages and limitations.

2.2. LDLR−/− Mice

The LDLR−/− mouse represents a model of familial hypercholesterolemia due to one of the mutations affecting the LDLR, and the plasma lipoprotein profile resembles that of humans. Mice, which are genetically deficient in LDLR manifested delayed clearance of VLDL and LDL from plasma. As a result, LDLR−/− mice exhibit a moderate increase of plasma cholesterol level and develop atherosclerosis slowly on normal chow diet [56]. Interestingly, the severity of the hypercholesterolemia and atherosclerotic lesions in LDLR−/− mice can be accelerated by feeding a high-fat, high-cholesterol diet [57], by mutating the apoB gene into an uneditable version [89], and by crossing either with leptin-deficient mice [10] or with apoB100 transgenic mice [11]. Under these conditions, the lesions in the aorta can progress beyond the foam-cell fatty-streak stage to the fibroproliferative intermediate stage.

In addition to LDLR−/− mice, the LDLR and apoE double-deficient mouse (LDLR−/−apoE−/−) which develops severe hyperlipidemia and atherosclerosis even on a regular chow diet, has been proposed as a suitable model to study the antiatherosclerotic effect of compounds without having to feed the animals an atherogenic diet [1213]. However, the response of both LDLR−/− and LDLR−/−apoE−/− mice to the treatment with hypolipidemic drugs varies from lowering of plasma cholesterol without atherosclerosis decrease to a weak lesion reduction with or without lower plasma cholesterol [414]. By contrast, those mice effectively respond to agonists of peroxisome proliferator-activated receptor (PPAR) or liver X receptor [1516]. This great variability indicates that LDLR−/− is probably not well-suited for analyzing the cholesterol-lowering and antiatherogenic effects of drugs.

2.3. ApoE−/− Mice

In 1992 two different groups simultaneously generated the apoE−/− mice by homologous recombination in embryonic stem cells [1718]. Homozygous deficiency in apoE gene results in a marked increase in the plasma levels of LDL and VLDL due to a failure in their clearance through the LDLR and LDLR-related proteins. The apoE−/− mouse contains the entire spectrum of lesions observed during atherogenesis and was the first mouse model described to develop lesions similar to those of human [1718].

Under normal dietary conditions, apoE−/− mice have dramatically elevated plasma levels of cholesterol, and they develop extensive atherosclerotic lesions widely distributed throughout the aorta [1820]. This process can be exacerbated on a high-fat diet, with female mice more susceptible than male mice [19]. A chronological analysis of atherosclerotic lesions in apoE−/− mice revealed that the sequential events involved in lesion formation in this model are strikingly similar to those in larger animal models and in humans. Predilection sites for atherosclerotic lesions in apoE−/− mice are the aortic root, followed by the aortic arch, the brachiocephalic trunk, the left carotid, and subclavian and coronary arteries [621]. Aortic lesions rapidly develop from initial fatty streaks comprised primarily of foam cells with migrating smooth muscle cells to more complex lesions in middle-aged mice. These advanced lesions are heterogeneous but typically composed of a necrotic core surrounded by proliferating smooth muscle cells and extracellular matrix proteins [2022].

The apoE−/− mice are currently the most widely utilized animal model for the study of atherosclerosis. In fact, the effect of many genes on the development of atherosclerosis has been examined by crossing the apoE−/− mice with other genetically manipulated animals. Furthermore, the apoE−/− mouse serves as a useful tool to: (i) identify atherosclerosis-susceptibility-modifying genes, by the candidate-gene and gene-mapping methods, (ii) decipher molecular mechanism and cell types involved in atherogenesis, (iii) search into the drug effects on atherosclerosis, and (iv) assess novel therapies that prevent lesion progression. In this sense, the apoE−/− mouse model was used to test additional therapeutic effects of statins beyond those attributable solely to cholesterollowering. One of the first observations was the paradoxical effect of simvastatin on atherogenesis in both apoE−/− and LDLR−/− mice [23]. In contrast to the atheroprotective effect of simvastatin in LDLR−/− mice, age-matched apoE−/− showed elevated serum total cholesterol and increased aortic plaque area, thus suggesting that the therapeutic effect of simvastatin may depend on the presence of a functional apoE [23]. In spite of this, the antiatherosclerotic effects of other statins have been effectively proven in apoE−/− mice [2425]. Several other compounds, such as angiotensin II receptor antagonists or PPAR agonists [26] also reduced the extent and severity of atherosclerotic lesions without lowering plasma cholesterol in apoE−/− mice. However, the recent finding of increased atherogenesis in apoE−/− mice treated with PPAR alpha and PPAR gamma agonists is consistent with clinical findings of the adverse cardiovascular events of dual therapy [27].

Nevertheless, a major limitation of the apoE−/− mouse model is the infrequency of plaque rupture and thrombosis, two common complications of human atherosclerosis. Ischemic cardiomyopathy has been occasionally found in aged mice [20], but interestingly, rapid coronary artery occlusion, myocardial infarction, and even premature death occur when apoE−/− mice were crossed with mice deficient in scavenger receptor class B type I or its adaptor protein [2829], thus mimicking many cardinal features of human coronary heart disease.

2.4. Transgenic Mice

Transgenic technologies have provided a series of very useful mouse models to study hyperlipidemia and atherosclerosis. Among them, mice expressing mutant forms of apoE, such as apoE3Leiden (E3L) and apoE (Arg 112→Cys→142) transgenic mice, are the more widely studied. These mice display a lipoprotein profile comparable to that of patients with dysbetalipoproteinemia, in which plasma total cholesterol and triglycerides are mainly confined to (V) LDL [30]. The E3L transgenic mice develop atherosclerotic lesions with all the characteristics of human vasculopathy, varying from fatty streak to mild, moderate, and severe plaques [3031]. Furthermore, E3L transgenic mice and the more recently developed E3L/Cholesteryl ester transfer protein (CETP) transgenic mice have been shown to be more sensitive to a variety of hypolipidemic drugs and PPAR agonists than apoE−/− and LDLR−/− mice [432].

2.5. Mouse Models of Diabetes-Accelerated Atherosclerosis

Diabetes is a high risk factor of cardiovascular disease. The cardiovascular complications of diabetes are manifested primarily as ischemic heart disease caused by accelerated atherosclerosis, and also as cardiomyopathy. Several models are available to study atherosclerosis and cardiomyopathy associated with diabetes, including apoE−/− and LDLR −/− mice in which type 1 diabetes is induced by streptozotocin or viral injection [3334]. In both mice, diabetes induction did not markedly change plasma lipid levels, thereby mimicking the accelerated atherosclerosis seen in patients with type 1 diabetes. Importantly, streptozotocin-injected atherosclerotic mice exhibited increased atherosclerosis in the aortic sinus, carotid artery, and abdominal aorta, as well as calcifications in the proximal aorta [3435].

In brief, mouse models have been very useful to unveil the importance of inflammatory and immunological mechanisms in the formation and progression of atheroma plaque. Recently, an enormous interest for the use of noninvasive magnetic resonance imaging (MRI) in mouse models of atherosclerosis has arisen [36], since MRI accurately characterizes the location, the size and the shape of lesions. In addition, MRI allows the differentiation between fibrous and lipid components of regress in plaques in mice. In combination with noninvasive imaging technologies, mouse models of atherosclerosis today also serve to test novel contrast agents, and to design and target specific molecules involved in high-risk plaque.

2.6. Rabbit Models

The high-cholesterol diet rabbit model has been widely used for experimental atherosclerosis. Back in 1913, cholesterol was found to cause atherosclerotic changes in the rabbit arterial intima, which is very similar to human atherosclerosis. Atherosclerotic lesions also develop in normolipemic rabbits as a result of repeated, or continuous intimal injury by an indwelling aortic polyethylene catheter, balloon angioplasty or nitrogen exposure. Therefore, many studies have used the rabbit model with high-cholesterol diet, arterial wall injury, or, most commonly, a combination of these two methods. In all these models, the observed lesions resemble, at least in part, those seen in human plaques, mainly regarding the inflammatory component, though the vascular smooth muscle cell proliferation determines for a great deal the lesion.

The rabbit model has largely been used to study the influence of lipid lowering (by diet or statins) on the plaque formation and “stabilization.” Those studies have contributed to unveil the mechanisms by which lipid lowering reduces macrophage accumulation and other aspects of atheroma inflammation [3738].

Recently, we have set up a novel rabbit model to examine the influence of inflammation on atherosclerotic plaque. The aim was to study some mechanisms by which atherosclerosis is particularly severe in patients with rheumatoid arthritis. Briefly, the model consists in a combination of femoral injury in hyperlipidemic rabbits and induced acute knee arthritis. Those animals had more intensive vascular lesions than animals without inflammation. This model could represent a novel approach to the study, inflammation-associated atherosclerosis [39].

A model for plaque rupture has been also developed in rabbits. Shimizu and coworkers [40] have developed a simple rabbit model of vulnerable atherosclerotic plaque, with the combination of aggressive vascular injury associated to a hyperlipidemic diet. The histological findings showed that an aortic plaque had the three features of “vulnerable plaque”: lipid-rich core, accumulation of macrophages, and a thin fibrous cap. In addition, a low-density lipoprotein (LDL) receptor-deficient animal model (the WHHL rabbit) has been developed. This model resembles to human familial hypercholesterolemia and shows evidence of progressive disease of the aorta with accumulation of birefringent lipids in intimal lesions and plaques, as well as in the media from birth to 1 year of age.

Although rabbit aortic arteries are smaller in vessel diameter than human carotid artery, they allows the studies with endovascular therapeutic devices. In addition, the rabbit model has also been used for the quantification of atherosclerotic aortic component by MRI. This technique accurately quantifies fibrotic and lipid components of atherosclerosis in the model and may permit the serial analysis of therapeutic strategies on atherosclerotic plaque stabilization [41].

2.7. Porcine Models

They prevention of heart attack and stroke depends on the detection of vulnerable plaques and development of plaque-stabilizing therapies. Animal models are essential for testing mechanistic hypotheses in a controlled manner, they should be representative of a human disease, and at the same time be easy to manipulate. However, vulnerable plaque recreation is one of the toughest tasks in animal model design. Plaque rupture is an additional complication of an already complex process, and the precise mechanisms involved remain hypothetical. A plethora of experimental approaches are available for growing atherosclerotic lesions in various animal species as mentioned above (Table 2).

tab2

Table 2: Animal models of plaque rupture and plaque associated thrombosis.

Currently, there is no single and golden standard animal model of vulnerable plaque, but pig models are probably the best way to recreate human plaque instability. The combination of diabetes and hypercholesterolemia constitutes a good model of accelerated atherosclerosis [42], and it was relevant study the role of certain biomarkers, such as the Lp-PLA2 since these animals share a similar plasma lipoprotein profile humans. In this regard, the selective inhibition of Lp-PLA2 by darapladib decreased progression to advanced coronary atherosclerotic lesions and confirmed a crucial role of vascular inflammation not associated to hypercholesterolemia, in the development of lesions implicated in the pathogenesis of myocardial infarction and stroke [43].

Several porcine models of advanced human-like coronary atherosclerosis have been employed to analyze the development and validation of coronary imaging technologies. In the evolving era of technological development, the availability and use of such animal models will become critical for the development of emerging technologies in interventional cardiology [44], and for the study of drug- eluting stents [45]. In addition, the porcine models of coronary atherosclerosis allow examining the impact of adventitial neovascularisation, on atherosclerotic plaque composition and vascular remodelling [46].

3. Animal Models of Abdominal Aortic Aneurysms (AAAs)

Animal models of atherothrombotic AAA are essential tools for the preclinical evaluation of new therapeutic strategies for the suppression of aneurysmal degeneration (Table 3). Recent insights into the mechanisms of human AAA have come from the studies in mouse models, and elastase-induced AAA in particular appears to recapitulate many features of human AAA. Here we briefly outline the most frequently used models of AAAs, and refer the reader to recent comprehensive reviews regarding additional animal models [4752].

tab3

Table 3: Current procedures for inducing AAA in animals.

3.1. Rat Models
3.1.1. Localized Aortic Perfusion with Elastase

This model consists of exposing a segment of the abdominal aorta and infusing it with elastase [53]. The degradation of elastic fibers triggers an inflammatory response that develops into an aneurysm [5455]. The severity of the induced AAA can be increased by adding plasmin to the infusion. This model has been adapted for use in several other species, including rabbit, mouse, and large animals.

3.1.2. Decellularized Xenografts

This model was based on the observation that chronic rejection of arterial allografts and xenografts, results in arterial wall dilatation and rupture. Michel and coworkers decellularized a section of abdominal aorta from one species (e.g., guinea pig), and the resulting tube of intact extracellular matrix was grafted into another morphologically compatible species, usually rat [58]. The xenogenic extracellular matrix triggers the destruction of host matrix, leading to aneurysm formation. The model has been successfully used to evaluate therapeutic targets [6469], although the heterogeneity of the aneurysms formed and the lack of vessel rupture are significant limitations.

3.2. Mouse Models

The mouse has become the preferred model for cardiovascular research for several reasons, including the ease of handling, low procedure costs, and the ability to manipulate the mouse genome. Consequently, of all animal models of AAA, mouse models have provided most of the insights into the mechanisms of human AAA. The following models are the most widely-used to date.

3.2.1. Calcium-Chloride-Induced AAA

In this method, initially developed in rabbits [62], calcium chloride is applied periaortically in the region between the renal arteries and the iliac bifurcation. Significant dilatation of the aorta is observed within 14 days, and the severity is significantly increased if calcium chloride is applied together with thioglycolate and if animals are fed a high-cholesterol diet [56]. Unlike other models, calcium chloride application induces AAA without the need for mechanical intervention.

3.2.2. Elastase-Induced AAA

The elastase-induced model was adapted for mice by Pyo et al. [57]. Elastase perfusion in mouse aorta causes a mild-to-moderate dilatation initially, which subsequently develops to a >100% increase in aortic diameter within 14 days. In this model, the degradation of medial elastin is delayed, and the subsequent aortic wall inflammation consists of mononuclear phagocytes throughout the adventitial and medial layers, with relatively few polymorphonuclear cells localized to the adventitia [57]. Elastase-induced injury increases the expression of MMPs, cathepsins, and other proteases [70], with MMP-9 being localized to aneurysm-infiltrating macrophages [71]. Elastase-induced AAAs thus appear to recapitulate many features of human AAAs, and this model has become a valuable and convenient tool for systematically evaluating the roles of individual gene products in aneurysmal degeneration [7180].

When compared to calcium-chloride-induced AAA, the main limitation of this method is in the mechanical stress required to recreate medial elastic degradation. However, the protocol resembles the time course of events leading to human AAA, including initial recruitment of leukocytes and mast cells, the development of a transmural aortic wall inflammatory response, and finally the upregulation of extracellular matrix metalloproteinases and other proteases, which induce a progressive degradation of the medial elastin and collagen, leading to the final aortic dilatation.

3.2.3. Angiotensin II-Induced AAA

This procedure was initially developed to define whether increased plasma concentrations of Angiotensin II (Ang II), have a direct effect on the atherogenic process in hyperlipidemic old apoE−/− mice. Unexpectedly, Ang II also produced large suprarenal abdominal aortic aneurysms in these animals [81]. In this model, inflammation of the vessel wall is associated with signaling through AT1a receptors [82], nuclear factor- (NF-) kappaB-mediated induction of proinflammatory genes, including MCP-1, M-CSF, iNOS, COX-2, inhibition of PPARs [83], activation of the NADPH oxidase p47phox [84], c-JUN N-terminal [85], Rho kinases [86], and enhanced recruitment of macrophages [8788] and extracellular matrix components and degrading enzymes [8991], leading to vessel dissection, and rupture. The severity of AAA is higher in hyperlipidemic apoE−/− or LDLR−/− male mice (~60% of mice), when compared to normolipidemic C57Bl/6 mice, although in these models neither hyperlipidemia per se nor atherosclerosis is considered major determinants [9294].

The model contributed to evidence the implication of the rennin-angiotensin (RAS) system in aneurysmal disease. However, two main limitations should be considered: the suprarenal location of the aneurysm (in contrast to the infrarenal location in humans) and the clinical relevance of RAS inhibition, since the association of RAS in human AAA has provided controversial results, pointing towards necessary large population studies.

3.2.4. Spontaneous Mouse Mutants

The blotchy mouse is a mouse strain containing a spontaneous mutation on the X chromosome which leads to abnormal intestinal copper absorption. These animals have weak elastic tissue due to failed crosslinking of elastin and collagen, and develop aortic aneurysms mainly in the aortic arch, thoracic aorta, and occasionally in the abdominal aorta [59]. However, results from this model are difficult to interpret, since the mutation produces many severe additional effects.

3.3. Rabbit Models

Several of the same interventions used in mice are also implemented in rabbits, including elastase infusion [60] and calcium chloride application to the abdominal aorta [56]. Another intervention used in rabbits is elastase infusion in the right carotid artery [61]. The main advantage of rabbits over other animal models is that rabbit aneurysms more closely resemble human aneurysms hemodynamically and histologically. Rabbit models also combine several of the attractive features of small animals, such as the easy housing and handling. In addition, similarly to large animals, rabbit aneurysms can be monitored by accessing through the femoral artery, thus providing an excellent model for testing endovascular therapies [9596].

3.4. Porcine Models

Porcine models of AAA have provided significant information about the changes that occur after AAA induction and about the responses to stent deployment. A recently-developed porcine model combines mechanical dilatation by balloon angioplasty with enzymatic degradation by infusion of a collagenase/elastase solution. The model is characterized by gradual AAA expansion associated with degradation of aortic wall elastic fibers, an inflammatory cell infiltrate, and persistent smooth muscle cell loss [63]. A broad number of similarities were found between this model and human AAA, and the procedure may also represent an excellent method to evaluate endovascular related procedures. Despite the benefits, however, pigs have significant disadvantages, including complex animal handling, the requirement of special housing and surgical room facilities, the elevated cost of the animals, and the reduced sample sizes per assay.

3.5. Thoracic Aortic Aneurysm (TAA)

Elastic tissue degradation is also related to the development of thoracic aortic aneurysm (TAA), and mouse models have significantly advanced the understanding of this pathology. TAA is a characteristic feature of Marfan syndrome (MFS), a disorder caused by mutations that affect the structure or expression of the extracellular matrix protein fibrillin-1, a glycoprotein that is associated of extracellular proteins, including integrin receptors and insoluble elastin [97]. Fibrillin-1 mutations in MFS decrease ECM sequestration of latent TGFβ, thus rendering it more prone to or accessible for activation [98100]. TAA progression in MFS is driven by elastic fiber calcification, vascular wall inflammation, intimal hyperplasia, structural collapse of the vessel wall, impaired activation of MAP kinase signaling, and altered synthesis of ECM proteins and matrix-degrading enzymes (MMPs) [101]. Systemic administration with TGFβ antagonists has been successfully used to mitigate vascular disease in mouse models of MFS and in children with severe and rapidly progressive MFS [97102]. Moreover, studies in mouse models have shown that fibulin-4 and LRP1 are also associated with TAA [103104].

4. Animal Models of Heart Failure

Models of heart disease in small animals, particularly rats, have been very useful for the assessment of pharmacological therapies. In addition, several target genes have been identified in genetically modified mouse models. Many of these genes have proved to be crucial in the initiation and progression of heart disease. Below, we describe the animal models currently used to study heart failure, which are also summarized in Table 4.

tab4

Table 4: Current procedures for inducing heart damage in animals.

4.1. Rat Models

Rat models have dominated research into heart damage because, while rats share many of the benefits of mice (low cost, ease of handling, etc.), their larger size greatly facilitates surgical and postsurgical procedures. Myocardial damage in rat hearts is induced by three procedures: surgical, pharmacological, or electrical.

The surgical method, first developed by Pfeffer and coworkers, consists of ligating the left coronary artery [105]. In this procedure, left thoracotomy is performed on the anesthetized rat, and the heart is rapidly exteriorized by gentle pressure on the right side of the thorax. The left coronary artery is either ligated or heat cauterized between the pulmonary artery outflow tract and the left atrium. The heart is then returned to its normal position and the thorax immediately closed. Several modifications have been introduced to improve performance and to reduce animal mortality, and left coronary artery ligation is the most common method used to induce acute myocardial damage in rat and other animal models. One important modification is temporary occlusion followed by reperfusion, allowing flow recovery through the previously occluded coronary artery bed. Left coronary artery ligation can thus be used to evaluate diverse parameters resulting from either permanent ischemia or ischemia/reperfusion.

Pharmacological induction of heart damage was first implemented by Bagdon and coworkers in 1963 and is achieved by treatment with the beta-one adrenergic receptor (B-AR) agonist isoproterenol [106]. Isoproterenol administration before ischemia exerts a cardioprotective action in rats, but at the right dose it induces cardiac myocyte necrosis and extensive LV dilatation and hypertrophy.

Isoproterenol treatment and left coronary artery ligation in rats are efficient and reproducible methods that provide valuable information about the underlying mechanisms implicated in human heart disease.

The electrical method consists of generating overlapping burns in exposed rat hearts by applying a 2-mm-tipped soldering iron to the epicardium of the left ventricle [107]. While this is also a valid method, the degree of heart damage produced is not consistent among laboratories, limiting the reproducibility of the results obtained with this procedure.

4.2. Mouse Models

Against the many general advantages of working with mice (ease of handling, low pregnancy times, etc.), investigators choosing them as models of heart failure must consider two important limitations: the small size of the heart and the structural differences with respect to the human cardiovascular system. Nonetheless, the availability of transgenic and knockout strains and the relative ease with which new genetic modifications can be introduced make the mouse one of the most attractive models for research into the molecular basis of heart failure.

One of the most widely used models of heart failure in mice is the left coronary artery ligation procedure, adapted from rat. In some protocols the artery is occluded permanently, but recently procedures for temporary occlusion have been introduced to reproduce human ischemia/reperfusion injury [108]. In this method the left anterior descending coronary artery is occluded and then reperfused, allowing flow recovery through the previously occluded coronary artery bed. Reperfusion is monitored visually, and the infarct can be analyzed by histopathological techniques, and can be documented in real time by non invasive high frequency. The areas at risk and the infarct size are revealed by staining with Evans blue dye and triphenyltetrazolium chloride and are assessed by computerized planimetry. This model has confirmed the benefits of reperfusion, since infarct size was found to be significantly lower than after permanent occlusion of the coronary artery.

The method has been further modified to analyze ischemic preconditioning of the heart. In this method, the left coronary artery is repeatedly occluded to subject the heart to several rounds of brief ischemia and reperfusion, followed by permanent occlusion. This approach has identified several ischemia-induced genes that confer tolerance to a subsequent ischemic event [122].

More recently, a model of myocardial infarction was developed, in mice and rats, in which a series of cryo-injuries is generated in the heart. This new model is yielding promising results [109].

4.3. Large Animal Models of Heart Failure

Small animal models have provided significant insights into human cardiac pathophysiology. However, rodent and human hearts differ in their architecture, heart rates, oxygen consumption, contractility, protein expression, and even stem cell populations, and there is therefore an obvious need for models of heart failure in large animals.

The first large animals used to study heart failure were dogs, in which models of myocardial infarction and serial microembolization of the coronary artery were developed [117]. However, the preferred large animal model of heart damage is the pig, because the collateral coronary circulation and arterial anatomy of pigs and humans are very similar and infarct size can be accurately predicted [123]. Among several models of MI in pigs, one of the most widely used is balloon occlusion of the left anterior descending coronary artery. In this model, a catheter is inserted through the femoral artery, positioning an angioplasty balloon over a guide wire at a position distal to the second largest diagonal branch of the artery, and infarction is induced by balloon inflation [120]. The similar size and cardiac physiology of pigs and humans mean that this model offers major advantages over models in other species. However, the method requires specialized equipment, dedicated surgical facilities and skilled personnel, limiting the number of laboratories able to conduct these studies.

The rabbit, much less expensive than pig, offers a compromise solution. Rabbit models of heart failure, including coronary artery occlusion models [114], have major advantages over other species. For example, the composition of sarcomeric proteins in rabbits is similar to that in humans, and the sarcolemmic reticulum contributes about 70% of calcium elimination. In addition, the WHHLMI rabbit strain provides a model of spontaneous myocardial infarction requiring no surgical intervention. This model was developed by selective breeding of coronary atherosclerosis-prone WHHL rabbits [124]. The main limitation of the WHHLMI model is that it does not feature plaque rupture, whereas in humans coronary plaque rupture and subsequent intravascular thrombosis are the major causes of acute myocardial infarction. Despite this limitation, the model is valid for the study of atherosclerosis-related heart complications [113125].

An additional model of heart failure in large and small animals is pressure overload of the left ventricle, induced by transverse aortic constriction in mice [110] and aortic banding in rats and rabbits. Left ventricle hypertrophy can also be recreated by ventricular pacing in dogs [115118126], valvular stenosis in rabbits [116], and renal artery constriction or aortic stenosis in rats, hamsters, mice, rabbits and dogs [111119].

Another model of heart failure is the dilated cardiomyopathy. Human dilated cardiomyopathy has been modeled in rabbits and pigs by inducing chronic tachycardia with a pacemaker [121127]. Transgenic mouse models, involving mutations that predispose to dilated cardiomyopathy, have also proved very useful. These models have identified an association of cytoskeletal and contractile proteins with this pathology, and very recently a transgenic model expressing a mutated cardiac alpha-actin gene was provided, in which calcium sensitivity of myofilaments is decreased and the expression of calcium/calmodulin-dependent kinase IIdelta (CaMKIIdelta) is increased [112]. Inhibition of CaMKII-delta in these animals prevented the increase in p53 and apoptotic cardiomyocytes and ameliorated cardiac function.

5. Conclusion

Animal models of cardiovascular disease yield important insights into the genetic basis of human cardiovascular diseases and provide a test bed for pharmacological and treatments. Nonetheless, investigators need to carefully consider their choice of model: no single method perfectly recreates the human disease, and there are related considerations of cost, infrastructure and the requirement for specialized personnel. Taking these considerations into account, experimenters therefore need to select models that best reproduce the aspect of disease being investigated. In particular, when moving from bench to bedside it is essential to test procedures in relevant models that yield highly reproducible results, but despite these limitations, given the range of animal models available today it will always be possible to devise an appropriate strategy, and animal models remain the best tools for advancing the understanding of the mechanism of human cardiovascular disease.

Acknowledgments

Authors’ work has been supported by Ministerio de Ciencia y Tecnología (SAF2007/63648, SAF2008/04629, SAF2009/11749, PI10/00072), CAM (S2006/GEN-0247), FIS (RECAVA RD06/0014/0035, PS09/00447), European Network (HEALTH F2-2008-200647), Euro Salud EUS2005-03565 and cvREMOD 091100. C. Zaragoza and C. Gomez-Guerrero contributed equally.

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Animal Models of Cardiovascular DiseaseModelos animales de enfermedad cardiovascular


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Journal Information Previous article | Next articleVol. 62. Issue 1.pages 69-84 (January 2009)Léalo en español Share PrintDownload PDF DOI: 10.1016/S1885-5857(09)71516-6Full text accessAnimal Models of Cardiovascular DiseaseModelos animales de enfermedad cardiovascularFrancisco J Chorroa, Luis Such-Belenguera, Vicente López-MerinoaServicio de Cardiología, Hospital Clínico Universitario de Valencia, Departamentos de Medicina y Fisiología, Universidad de Valencia, Valencia, SpainThis item has received18530VisitsArticle informationAbstract Full Text Bibliography Download PDFStatistics The use of animal models to study cardiovascular disease has made a substantial contribution to increasing our understanding of disease pathogenesis, has led to the development of diagnostic techniques, and has made it possible to verify the effectiveness of different preventative and therapeutic approaches, whether pharmacological or interventional. The main limitations stem from differences between human and experimentally induced pathology, in terms of both genetic regulatory mechanisms and factors that influence cardiovascular function. The experimental models and preparations used in cardiovascular research include those based on isolated cells or tissues or structures immersed in organ baths. The Langendorff system enables isolated perfused hearts to be studied directly under conditions of either no load or controlled loading. In small mammals, a number of models have been developed of cardiovascular conditions that result from spontaneous genetic mutations or, alternatively, that may be induced by specific genomic modification. One of the techniques employed is gene transfer, which can involve the controlled induction of mutations that result in the expression of abnormalities associated with the development of a broad range of different types of cardiovascular disease. Larger animals are used in experimental models in which it is important that physiological regulatory and homeostatic mechanisms are present.Keywords:Cardiovascular researchPreclinical researchAnimal modelsLa utilización de modelos animales para el estudio de enfermedades cardiovasculares ha contribuido sustancialmente al progreso en el conocimiento de su patogenia y ha permitido el desarrollo de técnicas diagnósticas y la validación de procedimientos preventivos y terapéuticos, tanto farmacológicos como intervencionistas. Las diferencias existentes entre la enfermedad humana y la inducida experimentalmente, tanto en los mecanismos de regulación genética como en los factores que determinan la función cardiaca y vascular, son sus principales limitaciones. Entre los modelos y las preparaciones empleados en la investigación cardiovascular, se encuentran los basados en la utilización de células aisladas y tejidos y estructuras en baños de órganos. El sistema de Langendorff permite el estudio directo del corazón aislado y perfundido aplicando diversas técnicas tanto sin someter al corazón a un trabajo como con una carga controlada. En mamíferos pequeños existen varios tipos de modelos de alteraciones cardiovasculares que ocurren por mutaciones genéticas espontáneas o son inducidos mediante modificaciones específicas del genoma. Entre los procedimientos utilizados se encuentran los basados en la transferencia genética con provocación controlada de mutaciones que dan lugar a la expresión de alteraciones asociadas al desarrollo de gran número de enfermedades cardiovasculares. Animales de mayor tamaño se emplean en modelos en los que se considera relevante que estén presentes los mecanismos de regulación y homeostasis del organismo.Palabras clave:Investigación cardiovascularInvestigación básicaModelos animalesFULL TEXT

GENERAL CONCEPTS

The use of animal models to study cardiovascular disease has substantially contributed to increasing our understanding of disease pathogenesis, leading to the development of diagnostic techniques as well as helping to verify the effectiveness of preventive and therapeutic procedures, whether pharmacological or interventional.1-8

Although animal models never completely resemble the clinical situation, they do make it possible to obtain direct information about specific events, offering good control over several variables while applying accurate, and typically invasive, procedures which are difficult to employ in clinical studies. The information obtained from animal models has to be assessed regarding its applicability to human pathology, and thus, information obtained in either context should be complementary.

Currently, animal experimentation is a legal requirement geared toward guaranteeing safety before introducing drugs and various diagnostic and therapeutic procedures into clinical practice. On the other hand, such experimental procedures are governed by a set of rules and regulations whose purpose is to avoid animals suffering during the development of experimental studies.9,10 It is compulsory to implement the means necessary to fulfill the established norms and approval procedures, and to monitor experimental protocols. To facilitate the development and acceptance of alternative methods of demonstrating specific pathophysiological or therapeutic hypotheses, the marketing of related techniques (eg, the in vitro production of substances based on animal models) and offering incentives for their use are measures that may help to develop conditions for experimenting with animal models.

The main advantages of these types of models lie in the use of control groups and setting the conditions that could modify the results after varying one or more factors (Figure 1). Some of the limitations stem from the differences between human and experimentally induced disease, such as differences in genetic regulatory mechanisms or in factors that influence cardiovascular function1 (Figure 2). There may be important anatomical differences between phylogenetically distinct species, they may respond to different pathophysiological mechanisms, and pharmacological treatment may act in different ways. For these reasons, the extrapolation of basic research findings to human pathology should always be undertaken with caution.

Figure 1. Main advantages of animal models used in the study of cardiovascular disease.

Figure 2. Limitations of animal models used in research on cardiovascular disease.

On the other hand, the gap between the laboratory and clinical practice sometimes means that the usefulness of the results of a specific study is not immediately apparent. To decrease this gap, intermediate steps should be created to bring the results of a basic discovery nearer to daily practice with the aim of preventing, diagnosing, or treating cardiovascular disease. Basic research scientists also need to know about the concerns arising from clinical practice, unsolved problems, or observed limitations, in order to identify gaps and try to guide basic research efforts toward solving these problems. Science-based communication and discussion within multidisciplinary teams is essential to improving research findings that, in the final analysis, should be aimed at better disease control and promoting health in our society.

TYPES OF ANIMAL MODELS

The application of surgical procedures, controlled administration of drugs and compounds, selection of spontaneous genetic mutations and, more recently, genetic modification through recombination procedures have provided a large number of models with which to study cardiovascular disease. The anatomical and pathophysiological substrata may differ according to the animal species used, and different models can vary in their response to experimentally induced modifications.11

The appropriate selection of the experimental model should be based on the purpose of the study, that is, the model that is best suited to the research aims, although various factors may influence this decision, such as the techniques and methods required to obtain the information, their availability and accessibility, the conditions required to house the animals, the number of experiments, the type of study, acute or chronic, and in the latter case, its duration. Some of the models and preparations used in cardiovascular research are presented below.

Isolated Cells and Cell Cultures

Different techniques are available to isolate cardiac myocytes from different animal species. At its simplest, the procedure consists in isolating the heart, perfusion with calcium-free enzyme solutions to degrade the collagen matrix connecting the myocytes, followed by cell extraction.4,12 Once cell viability is verified, various study techniques can be applied, eg, electrophysiological techniques, such as patch-clamping, to analyze ionic currents or changes in transmembrane voltage (Figure 3).

Figure 3. Diagram of the use of the patch-clamping technique in isolated cells with the intact membrane (upper part), after perforating the membrane (middle part), or using fragments of cellular membrane (lower part).

The development of cell culture techniques in combination with those of genetic engineering has increased the possibility of analyzing various events.5,13-17 Thus, transfection techniques, in which genetic material (recombinant DNA) is introduced into the nucleus of various mammalian cells, enable protein expression to be modified and, thus, cell functions. Studies which have used these techniques include those aimed at analyzing the behavior of the ionic channels involved in the kinetics of transmembrane action potential under basal conditions and under the action of various drugs.14-16

In other areas, such as regenerative medicine and tissue engineering, multiple lines of research are being developed with the aim of understanding and controlling the process of cellular differentiation. Among the possible uses are the development of tissue structures with potential clinical application.18

Tissue and Organ Bath Preparations

The study of specific structures from various animal species, such as papillary muscles, Purkinje fibers, myocardial tissue layers, or arterial and venous vessels, provides information in various fields that range from cardiac and vascular mechanics to electrophysiology.

Papillary muscle preparations obtained from small animals are based on extracting muscle and placing it in a chamber through which liquid nutrient flows at a constant temperature and that makes it possible to attach stimulation electrodes and force transducers to the ends of the muscle. Various mechanical properties can be studied in these preparations, such as the force-frequency relationship, responses to different types of overload and the mechanisms that regulate them, as well as the effects of various pharmacological or non-pharmacological manipulations.19,20 The use of microelectrodes or extracellular electrodes has enabled the study of the electrophysiological properties of myocytes and the analysis of drug effects,21 for example, their capacity to prolong repolarization at different frequencies.12 The use of these techniques in genetically modified models has increased our capacity to analyze the pathophysiological processes involved in various human diseases.

In addition, organ baths can be used to study myocardial layers, vascular rings, or various preparations that include the sinus node, the atrioventricular node or Purkinje fibers. Access to the elements constituting the preparation makes it possible to obtain relevant information on its behavior.22-24 One of the drawbacks of these tissue and organ preparations lies in the possible difficulties involved in the adequate dissemination to their deepest layers of oxygen, solutes or drugs and the substances under study.25,26 This limitation is not an issue when dealing with isolated myocyte preparations.12 The use of more complex experimental procedures can also be avoided,27 in which the part of the heart to be studied is isolated (eg, the right atrium with the sinus node or the papillary muscle with the interventricular septum) and irrigated with blood from the arterial system of another animal (cross circulation) or with liquid nutrient through the coronary arteries that replace the circulation to these tissues (eg, the sinus node artery or the anterior septal artery).Langendorff System (Isolated Heart)

The Langendorff method for studying isolated heart was described at the end of the 19th century. This method allows us to study events without neurohumoral interference, under controlled conditions, and with direct access to the areas of interest (Figure 4). Myocardial perfusion is done via the coronary arteries that are filled in a retrograde manner. In the classic preparation, the heart contracts without performing work, although variants exist (working heart) where, through various devices, the heart contracts under a specific load (liquid-filled intraventricular balloon, perfusion from the left atrium with the aorta connected to a system that regulates flow resistance, etc).2,28 Myocardial ischemia can occur after coronary artery ligation or by reducing liquid nutrient oxygenation. Hearts are used from different species, ranging from rat to large animals. After the heart has been extracted, the sectioned portion of the ascending aorta is cannulated and liquid nutrient supplied through this, via the sinuses of Valsalva to the coronary arteries, irrigating the myocardium and draining to the exterior through the cardiac venous system. The perfusion pressure (between 60 mm Hg and 80 mm Hg) or flow (between 1.5 mL/g and 2.5 mL/g) are kept constant and the liquid nutrient is oxygenated with a mixture of O2 (95%) and CO2 (5%). This liquid contains suitable proportions of electrolytes and glucose (Krebs-Henseleit solution, Tyrode solution, etc) and can be complemented with albumin or blood, especially when hearts from large species are used. The pH is kept between 7.35 and 7.4 and temperature is normally kept between 36oC and 37oC.

Figure 4. Diagram the Langendorff system used to study isolated perfused heart preparations from different animal species.

Multiple extracellular electrodes—both epicardial and endocardial or intramyocardial—can be used in the isolated perfused heart to conduct mapping studies of cardiac electrical activity and to analyze basic electrophysiological properties (refractoriness, conduction, automatism), their modification through the use of drugs, physical agents, electrical stimuli, or other procedures, as well as the mechanisms of onset, perpetuation or termination of cardiac arrhythmias29-33 (Figures 5-8). If the isolated heart is submerged in serum saline or in the liquid nutrient itself, the equivalent to electrocardiographic traces can be recorded and their variations analyzed under different maneuvers or drugs. The use of suction or pressure electrodes makes it possible to obtain the so-called monophasic action potentials which are related to the characteristics of transmembrane action potentials.34 The use of optical mapping systems based on recording changes in marker fluorescence associated with variations in membrane potential (Figure 9) has made it possible to approach the study of cardiac arrhythmias, providing additional information on the duration of action potential.35-37

Figure 5. Various types of extracellular electrodes used to record myocardial activation processes in isolated perfused rabbit heart.

Figure 6. Devices used to conduct mapping studies of cardiac electrical activity by introducing thermal variations in isolated perfused rabbit heart.

Figure 7. Upper part: activation map obtained with a multiple epicardial electrode, located on the anterior wall of the left ventricle in an isolated perfused rabbit heart preparation during epicardial stimulation. Lower part: identification of the ischemic area after coronary occlusion by analysis of ventricular repolarization modifications.

Figure 8. Frequency domain analysis (spectrum and time-frequency analysis) of the signals recorded during ventricular fibrillation in the left anterior ventricular wall. Isofrequency maps are shown at the lower left.

Figure 9. Images obtained using an optical mapping system during a ventricular fibrillation episode through recording changes in marker fluorescence in relation to variations in membrane potential.

Small Mammals

When choosing and using models based on small animals the particular characteristics of each species have to be taken into account; for example, collateral circulation is highly developed in the coronary tree of guinea pigs and hence ligation of one of the main branches would make it impossible to stably reproduce situations of myocardial ischemia. Among other advantages related to the use of these types of animals are their high reproduction rate (important when selecting cases with specific genetic characteristics within a suitable time-frame) and short mean lifespan, and thus the natural history of the disease can be analyzed within a brief period. On the other hand, genetic similarity to humans enables the study of specific alterations and their correspondence to diseases found in clinical practice, and this provides more information on the defects implicated in inherited diseases. Nevertheless, differences between the basic mechanisms that may restrict extrapolating the results have to be taken into account.1-4

In small mammals, several types of models have been developed of cardiovascular abnormalities that occur spontaneously or that are induced experimentally.3 The potential for research is increased by having a large number of animals available through selective breeding. Mouse models based on spontaneous genetic mutations and the development of strains with polygenic or monogenic mutations have facilitated the study of the mechanisms of various pathophysiological processes and their genetic determinants. On the other hand, in animals such as the mouse, technological advances have made it possible to create models with mutations in specific genomic loci. These procedures range from non-transgenic ones, based on induced mutations and the development of strains obtained from different types of embryos, to those of gene transfer, that were begun more than 25 years ago and that have given way to controlled induced mutation procedures with the expression of mutations associated with the development of a large number of diseases.3,5,7,38,39

Transgenic models are based on introducing DNA into the genome of a specific animal, a fact made possible following the development of technology used in cell cultivation, manipulating embryos, and DNA recombination. Directly related techniques include pronuclear injection, viral transgenesis, and homologous recombination. Pronuclear injection involves the microinjection of DNA into the pronucleus of the zygote, and in viral transgenesis viral vectors are used to introduce genes such that they are expressed or alter the genome. Recombinant techniques are used to introduce a particular alteration into a specific site of the genome. In these cases, genetic mutation is obtained by specific genetic modification. Thus, in so-called knockout mice (genetic inactivation by homologous recombination), particular genes are inactivated to obtain information on their function, thus enabling the study of diseases created by mutations where there is a total or partial loss of protein formation through those which the gene expresses.3 On the other hand, in so-called knockin mice, specific genes are replaced or mutated, and their expression patterns are analyzed as well as the effects of these variations that involve a functional change.3,39

The procedure for creating these models is based primarily on the manipulation of embryonic stem cells. These are obtained from the inner cell mass of male blastocysts after egg fertilization and, once cultivated, specific mutations are created in vitro. These cells are pluripotent and are used to create mice with chosen mutations. Mutations are induced by introducing previously constructed genetic material into the embryonic cell. To this end, the electroporation procedure is used (via applying an electrical field), after which a small proportion of the cells undergoing the process incorporate the previously designed and constructed modifications. After multiplication of the embryonic cells carrying the mutation (heterozygous cells), these are injected into previously stored blastocysts which are later implanted in pseudopregnant female mice. Their chimeric offspring, which are derived from the modified embryonic cells and the donor blastocysts, are identified and after the heterozygous mice are crossbred, homozygous mutant mice are obtained (Figure 10).

Figure 10. Genetic modification procedures through specific gene inactivation, replacement or mutation. These are based on the manipulation of embryonic stem cells obtained from blastocysts and in which specific mutations are created by introducing previously constructed genetic material. After multiplying the embryonic cells carrying the mutation (heterozygous), they are injected into blastocysts that are later implanted in female mice. After the crossing the chimeric heterozygous offspring, homozygous mutant mice are obtained.

Lipid metabolism has been modified in transgenic rodent models to approach that of humans in order to produce models of atherosclerosis.40,41 Models of heart failure,42-44 several types of cardiomyopathies,7 and genetically-induced arrhythmias have been developed that enable the identification of forms of expression and the analysis of related genes.2,5-6,38,39 Thus, arrhythmic syndrome models are studied with characteristics common to those found in humans. For example, regarding atrial fibrillation, some of the transgenic models are characterized by the development of atrial fibrosis, or connexin 40 knockout models are created in which there are conduction abnormalities. Models have also been created in which the myocardial electrophysiologic properties are altered, with accelerated repolarization, such as occurs in those overexpressing Kir2.1 protein, altering the inward rectifying potassium current (IK1).6 Various models have been developed in relation to ischemic heart disease, including those used to study ischemic preconditioning and its relationship to connexin 43 and gap junctions.45,46

Larger Mammals

Large animals models are used in which it is important that regulatory and homeostatic mechanisms are present. Various aspects can hinder the interpretation of the results and these have to be taken into account when proposing and designing studies.2 Differences exist between species; for example, in the pig, the coronary circulation is characterized as terminal, without anastomoses between vascular branches, whereas in the canine model collateral circulation is present (Figure 11). Thus, coronary ligation in the pig resembles coronary occlusion in subjects without collateral circulation, but is unlike that of patients who have developed occlusion either spontaneously or in the context of chronic ischemic heart disease. On the other hand, there also are differences between species in the relative importance of the different branches of the coronary tree in relation to the size of the territory they irrigate. Regarding the cardiac conduction system, the distribution of the Purkinje fibers is not uniform; in the pig this is transmural, ranging from the endocardium to the epicardium,2

Figure 11. Characteristics of the collateral circulation in different animal species (adapted from: Maxwell MP, Hearse DJ, Yellon DM. Species variation in the coronary collateral circulation during regional myocardial ischaemia: a critical determinant of the rate of evolution and extent of myocardial infarction. Cardiovasc Res. 1987;21:737-46).

whereas in the dog or cat this is predominantly subendocardial, more closely resembling that of the human, thus presenting a more similar ventricular activation sequence. Differences exist between the myocardial repolarization process in canine models and humans, making it difficult to compare the changes occurring at the QT interval or T wave under different circumstances. Other differences between species stem from ion channel expression which regulates the characteristics of myocardial cell action potentials, since in some species, such as rats, the IKr channels are not expressed, whereas they are expressed in rabbits, guinea pigs, and dogs, and this fact aids in studying certain events such as arrhythmias associated with the prolongation of the QT interval.

Another aspect to take into account when using these experimental models is choice of the appropriate form of anesthesia, since its effects can modify the events studied.2 Pentobarbital effects the parasympathetic nervous system and influences the distribution of intramyocardial blood flow, whereas the combination of morphine with a-chloralose has less effect on autonomic control of the cardiovascular system, although it prolongs ventricular repolarization. Short-acting anesthetics can be of use in the initial stages of specific protocols where anesthesia is later maintained with other drugs. The anesthetic regimen can influence to what degree sedation is stable and some of their effects should be taken into account, such as depressed contractility or modifications in autonomic tone (pentobarbital, propofol). Certain inhaled anesthetics can modify the electrophysiological properties of myocardial cells and can have proarrhythmic effects (halothane, enflurane).

Chronic experiments range from those based on an acute intervention after which the effects produced by this in the presence or absence of specific drugs are observed (such as modifications in the degree of myocardial fibrosis after inducing atrial overload by valvular dysfunction) to those that require the use of special equipment to monitor the variables under study through different procedures, such as telemetry or cable transmission, and that, as a result, require the implantation of various systems (Table 1).

ANIMAL MODELS IN DIFFERENT AREAS OF CARDIOVASCULAR RESEARCH

Atherosclerosis

A large number of processes associated with atherosclerosis have been studied in animal models.40 Rabbits fed with cholesterol-rich diets have been studied to analyze lesion formation and progression, as well as lesion regression after the diet has been modified,47 or when specific treatments are added,48-51 and which have also been assessed in other animal species.52-56 In rodents, the application of transgenic techniques has enabled the creation of models with the lipid metabolism abnormalities present in humans,57 for example, through the manipulation of apolipoprotein E expression.58 In these mice, gene transfer using vectors that contain apoA-I cDNA to increase the concentration of high-density lipoprotein cholesterol (HDL-C) has led to lesion regression,59 and this has also been achieved by prolonging apolipoprotein E expression through the use of viral vectors.60,61 The transplantation of aortic segments that contain atherosclerotic lesions from transgenic mouse models with high apolipoprotein B levels, low HDL-C concentrations, and fed with hyperlipidemic diets to normal recipients has enabled the study of the processes involved in the plaque stabilization and regression.62,63 Phenomena such as thrombogenesis and platelet function have also been analyzed in animal models,64 where the use of purpose-built perfusion chambers65 has enabled the analysis of the mechanisms involved in thrombosis.

Ischemia-Reperfusion and Myocardial Infarction

Various lines of research in animal models have focused on analyzing the potential to limit the size of the infarction. Some decades ago, studies were conducted on drugs and substances that could slow down or prevent the development of necrosis,66,67 with limited and nonreplicable results.46 However, experimental models of coronary occlusion provided accurate data on the development of necrosis.68-73 After the relevance of the role reperfusion played in saving ischemic myocardium was recognized, its implication in lesion development also began to be analyzed. Pathological mechanisms associated with calcium overload, the action of oxygen free radicals, and the activity of inflammatory cells with the involvement of leukocytes, adhesion molecules, and cytokines, among other factors, have been identified in animal models.74 Mitochondrial damage has also been analyzed75 by identifying membrane abnormalities (pore formation) that make it permeable to various substances and ions and that determine cell death.

The inhibition of pore-opening may be associated with cardioprotection during ischemia-reperfusion.

Progress has also been made in the areas of ischemic preconditioning and postconditioning.76,77 Experimental protocols exist with interventions that replicably limit the size of the infarction due to preconditioning; for example, in canine models, this can be achieved by occluding the anterior descending coronary artery for several 5-min periods, separated by similar periods during which perfusion is reestablished, and after which prolonged occlusion is established. Postconditioning also reduces the size of the infarction; for example, by the application of several intermittent short-term periods of coronary occlusion (30 s) at the beginning of reperfusion following prolonged occlusion. Both phenomena have been studied in different animal species and the time course of the protective effect and molecular mechanisms involved have been analyzed, and which continue to be subjects of research.46,78-84

Cardiomyopathies

Dilated cardiomyopathy, which is characterized by ventricular dilatation and deterioration in systolic function, is one of the major causes of heart failure as well as being the most frequent cause. Among studies analyzing its pathogenesis are those focusing on familial forms, associated with genetic mechanisms, such as cardiac a-actin, the b-myosin heavy chain or troponin T, and that lead to various abnormalities.

Models have been developed in small animals, such as rats, mice, and hamsters in order to study its pathogenesis.7,42,85,86 In hamster strains with spontaneous dilated cardiomyopathy (BIO14.6, CHF 147) the steady development of rhabdomyolysis, hypertrophy, dilatation, and congestive heart failure has been described, with changes in enzymatic expression that lead to an increase in oxidative stress (TO-2 strain). In genetically modified models,42,86 dilated cardiomyopathies have been induced that include mutations in the genes that regulate the cytoskeleton (lack of genes for dystrophin and utrophin) and cause muscular dystrophy and cardiomyopathy. Muscle LIM protein knockout models have also been created that develop dilated cardiomyopathy. G protein overexpression leads to cardiomyopathies in which apoptosis mechanisms are involved. Mitochondrial mutations exist with deficits in oxidative phosphorylation occurring with dilated cardiomyopathies. In large animal models, closer to clinical practice, aspects associated with mechanics, molecular and metabolic abnormalities, and inappropriate neurohumoral adaptations have been analyzed.

Heart Failure

The analysis of the causes of heart failure, and the transfer of existing knowledge from experimental studies to clinical practice—both in the area of myocardial protection and ventricular remodeling—form another challenge that faces the researcher studying cardiovascular disease.

The development of heart failure involves both the heart and peripheral circulation and the complex neurohumoral mechanisms that regulate the hemodynamic processes. Small-animal strains exist in which heart failure has appeared due to spontaneous genetic alterations. Animals in which genetic modifications have been artificially induced are also used.43,44,86 Among the murine models where transgenic and knockout technology has been applied,43,44 are those which are characterized by abnormalities in calcium regulation and the development of hypertrophy.42,86 In these models, studies focus on the relationship between the expression of sarcoplasmic reticulum Ca2+ ATPase (SERCA) and the development of heart failure after its expression has been altered.

In larger animals, various procedures can be used to produce heart failure models, among which are rapid atrial or ventricular stimulation (3-4 times higher than spontaneous sinus rhythm),87 that within a period of 3-5 weeks causes heart failure with reduced systolic function, reduced cardiac output, increased systemic vascular resistance, increased systolic ventricular wall stress and electrophysiological and metabolic abnormalities. Various types of pressure or volume overload or myocardial ischemia are also used; for example, by provoking valvular failure leading to volume overload or by increasing ventricular ejection impedance leading to pressure overload. In addition to analyzing the factors that determine deterioration in ventricular function in these models, the response to various treatments are analyzed and various circulatory assistance techniques are assessed.

Arrhythmias

It is difficult to find models of arrhythmia that bring together all the determining anatomicopathological, electrophysiological, biochemical, and molecular factors that are present in clinical practice. Transgenic models are used to study the mechanisms involved in the genesis of several types of cardiac arrhythmias in the context of channelopathy and various types of cardiomyopathies.5,38,39 Animal models have contributed relevant information on the mechanisms of atrial fibrillation and various therapeutic procedures.6 There are several models of atrial fibrillation in different contexts which provide complementary information: vagal nerve stimulation or continuous perfusion with acetylcholine, persistent overstimulation, congestive heart failure, sterile pericarditis, atrial ischemia, mitral regurgitation, volume overload, respiratory failure, drug action (cesium to induce the formation of afterpotentials and abnormal automatisms, and aconitine to create ectopic foci) and transgenic models, among others.6

In the context of myocardial ischemia and reperfusion, various types of arrhythmias have been generated.46,88,89 Their analysis has contributed relevant information on sudden death, for example. One of the most frequently used animal models to study these events is the two-stage Harris model, where the anterior descending coronary artery is ligated with a hypodermic needle placed parallel to the artery. The needle is immediately removed to prevent complete occlusion, while producing a stenosis and a consequent reduction in flow; 30 min later the artery is completely occluded by a second ligature.2,90,91 This procedure—or those based on it that use ligatures to achieve a degree of obstruction controlled through the analysis of distal flow— prevents the immediate development of ventricular fibrillation due to ischemic preconditioning92 and thus various events produced by the myocardial ischemia, both mechanical and electrical, can be studied. These models have helped to determine the key role of abnormal automatism of partially depolarized Purkinje fibers during the onset of arrhythmias sustained during the first hours after coronary occlusion.89 It has also been observed that activity triggered by afterpotentials is a later event. Various models have been developed in order to better understand the events that occur in chronic ischemic heart disease.93 Simultaneous ischemia and physical exercise can help in understanding the mechanisms involved in the onset of arrhythmias in these situations, both without previous infarction and following infarction, as well as the factors that determine or facilitate their appearance or the possible beneficial effects of therapeutic intervention, such as those designed to prevent ventricular fibrillation and sudden death. Autonomic nervous system activity triggered by reflexes initiated by ischemia, hemodynamic changes produced by ischemia, or simultaneous exercise has been studied from various standpoints, including analyzing the intensity of the baroreceptor response that determines the probability of ventricular fibrillation occurring.93,94

The induction of specific arrhythmias can also be studied using programmed stimulation techniques. Other methods of inducing arrhythmias2 are based on the use of drugs or substances that facilitate the production of afterpotentials (catecholamines, digitalins),95 those that generate repetitive activity (ectopic foci, aconitine), or that simulate or promote the production of arrhythmias associated with prolonged QT intervals (torsades de pointes) (antiarrhythmic drugs with an inverse effect depending on frequency or other types). Clofilium, which blocks HERG channel conduction, or methoxamine, can cause torsades de pointes in rabbits in vivo. Veratridine in isolated perfused guinea pig heart2 or bradycardia induced by complete atrioventricular blockage in large animals—leading to dilatation and ventricular hypertrophy—are other models used to induce polymorphic tachycardias. Parameters that predict arrhythmias have been analyzed in these models, such as those related to QT interval variability and also the proarrhythmogenicity of specific drugs before their introduction into clinical practice.2

The concept of electrical and structural remodeling in atrial fibrillation has been based on experimental studies96-98 and has led to some therapeutic interventions being reassessed. Changes in refractoriness, conduction velocity, and the wavelength of the activation process associated with the development time of the arrhythmia have been studied in this arrhythmia,99 in addition to the ionic mechanisms involved, and changes associated with structural remodeling characterized by the development of fibrosis and conduction abnormalities. Various therapeutic approaches aimed at controlling atrial fibrillation have benefited from data obtained from animal models, including information on causal mechanisms and responses to pharmacological or surgical interventions or those based on radiofrequency ablation procedures.100-106 It has been verified that specific drugs stop arrhythmia without modifying the wavelength of the myocardial activation process.107

Controlling sudden death due to ventricular arrhythmias remains an unsolved problem and a challenge for researchers working in various disciplines. The clinical outcomes of the use of implantable automatic defibrillators are an example of progress in this field and experimental studies have contributed to their development.108

FUTURE DIRECTIONS

Progress in cardiovascular disease control is based on improved understanding of a range of highly varied issues, from those addressed in epidemiological studies, clinical trials, and registries to those studied within basic research projects. In this field, progress has been made in molecular analysis: the genetic determinants of a large number of processes, their interactions, transcription mechanisms or signaling pathways, have all provided new insights into understanding the development and functions of organs and systems.

The use of classic techniques, such as the Langendorff preparation, and variants where the heart undergoes controlled loading, as well as genetic modification procedures, have furthered the study of cardiac mechanics and contraction and relaxation processes. This has enabled analysis of the functional effects of abnormalities in the expression of proteins associated with sarcoplasmic reticulum, intracellular calcium regulation or elements of the cardiac contractile apparatus.109 In vivo determinations in murine models with genetic abnormalities that produce hypertrophy contribute information on the factors that determine systolic and diastolic function and on the pharmacological interventions that modify them. The use of these models requires adapting the recording systems to the characteristics of small transgenic animals.

The introduction of non-invasive techniques, such as echocardiography, with high-resolution transducers (high-emission power and better temporal resolution to adapt it to the high heart rate in these animals), has made it possible to obtain more information on anatomy and cardiac function and study the results of pressure and volume overloads in transgenic models, and thus analyze the factors associated with the development of hypertrophy or ventricular dilatation. The use of intracavitary catheters combined with ultrasound techniques has aided the analysis of pressure-volume curves.

More recently, cardiac magnetic resonance imaging has been introduced into experimental studies to obtain anatomical and functional information, which, together with ultrasound techniques can be applied in vivo. It also contributes information on perfusion and myocardial metabolism, the presence of myocardial necrosis, and on calcium regulation during the stimulation-contraction processes.110 Tools are being developed to study the mechanics of intramyocardial function and torsion. Metabolic analysis includes spectroscopic methods for detecting P31 or H1, establishing ratios such as that of phosphocreatinine and ATP, associated with energy production. It also enables the analysis of the basic mechanisms that influence cardiac function, ventricular remodeling processes, and postinfarction scar formation. The study of new agents which make the controlled analysis of the myocardium possible enables the assessment of various events such as myocardial cell apoptosis.

Positron emission tomography is being used in experimental models to analyze and quantify changes in glucose in the myocardium and to detect regional perfusion defects and, together with single-photon emission computerized tomography (SPECT), contributes molecular information through the use of markers that bind to specific targets.

In the area of cardiac electrophysiology, optical systems have been refined to the point of obtaining temporal resolutions comparable to those obtained with extracellular electrodes, thus extending the study of cardiac arrhythmias. This also involves progress in the development of microelectrode arrays capable of stimulating and recording bioelectrical signals with high temporal and spatial resolution, making it possible to accurately study the interactions and linkages between different types of cells and tissues, as well as to determine the effects of drugs on the characteristics of action potentials.111

This work was partly conducted with research grants from the Ministry of Health PI06/0758 (Project FCI) and RD06/0003/0010 (RETIC: REDINSCOR).


Correspondence: Dr. F.J. Chorro.
Servicio de Cardiología. Hospital Clínico Universitario.
Avda. Blasco Ibáñez, 17. 46010 Valencia. España.
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CLINICAL PUZZLEHeart failure and mouse modelsRoss BreckenridgeDisease Models & Mechanisms 2010 3: 138-143; doi: 10.1242/dmm.005017

Abstract

Heart failure is a common, complex condition with a poor prognosis and increasing incidence. The syndrome of heart failure comprises changes in electrophysiology, contraction and energy metabolism. This complexity, and the interaction of the clinical syndrome with very frequently concurrent medical conditions such as diabetes, means that animal modelling of heart failure is difficult. The current animal models of heart failure in common use do not address several important clinical problems. There have been major recent advances in the understanding of cardiac biology in the healthy and failing myocardium, but these are, as yet, unmatched by advances in therapeutics. Arguably, the development of new animal models of heart failure, or at least adaptation of existing models, will be necessary to fully translate scientific advances in this area into new drugs. This review outlines the mouse models of heart failure in common usage today, and discusses how adaptations in these models may allow easier translation of animal experimentation into the clinical arena.

Heart failure: the medical problem

Heart failure is an increasingly common diagnosis, with a dismal prognosis that is worse even than many types of cancer (Ho et al., 1993). There are few therapeutic options. The estimated cost of heart failure is currently between 1–2% of the total healthcare spend in developed economies and is expected to rise (McMurray and Stewart, 2000).

Clinical presentation may be insidious or acute, with decreased exercise tolerance and shortness of breath. Cardiac arrhythmias may accompany heart failure, leading to high rates of sudden death. Current treatment includes simultaneous administration of angiotensin-converting enzyme (ACE) inhibitors (acting principally by vasodilatation), β blockers (slowing the heart rate) and spironolactone (vasodilatation and diuresis). This combination reduces death resulting from heart failure, but these drugs do not ‘cure’ the condition. There are no truly ‘disease modifying’ drugs for heart failure.

Many conditions eventually lead to heart failure (Table 1), several of which are associated with each other, such as hypertension, obesity and diabetes, as exemplified in the case study. Our understanding of the heart failure disease phenotype is not yet sufficient to appreciate whether aspects of the final heart failure syndrome differ with the causative aetiologies. Preliminary evidence, however, demonstrates that different gene expression patterns (Huang et al., 2005) and prognoses (Felker et al., 2000) are associated with heart failure resulting from differing causes.

Heart failure comprises changes in cardiac contractility, electrical conduction and energy metabolism, leading to an inability of the heart to meet circulatory demands (Jessup and Brozena, 2003Stanley et al., 2005). This activates neurohormonal compensatory mechanisms, such as vasoconstriction, which are thought to be helpful in maintaining general organ perfusion in the short term, but maladaptive with respect to long-term cardiac function. In this way, the physiological and gene expression changes observed in heart failure are not constantly ‘adaptive’ or ‘maladaptive’ (and are, thus, not easily amenable to pharmacological modulation). This has introduced therapeutic confusion over the years. For example, use of β-adrenoceptor antagonists was discouraged in heart failure for many years on the basis of studies showing increased mortality when given to acutely unwell patients (Braunwald and Chidsey, 1965). Subsequently, β blockers have been found to be beneficial when used in relatively low doses in stable heart failure patients (CIBIS, 1999).

An increasingly commonly recognised variant of heart failure is ‘diastolic’ or ‘systolic function preserved’ heart failure, characterised by resistance to ventricular filling rather than defective contraction (Dodek et al., 1972Zile et al., 2004). As the commonest causes of diastolic heart failure are ischaemia, obesity, hypertension, diabetes and ageing (Owan and Redfield, 2005), the incidence of this condition is expected to increase with time. Fundamentally, the mechanisms underlying this variant of heart failure are unknown.

There have been major recent advances in the clinical assessment of heart failure patients – the aim being earlier diagnosis and risk stratification (i.e. the identification of high-risk patients). Clinical scoring based on a patient’s assessment of their own exercise capacity and basic clinical observations has proved of use in identifying high-risk patients and guiding therapy (Goldman et al., 1981). More recently, imaging technology has been used to improve diagnosis and prognostication in heart failure. Echocardiography has been the historical ‘gold standard’ for non-invasive evaluation of the failing heart, and is safe and relatively cheap. The recent development of tissue Doppler measurement to evaluate myocardial strain has improved sensitivity with respect to early detection of abnormalities. Further adaptations, such as ultrasonographic tracking of acoustic markers (‘speckle tracking’) and intravascular ultrasound contrast (Flu et al., 2009), have shown promise at their relatively early stage of development. Cardiac magnetic resonance imaging (cMRI) is increasingly used in heart failure and, as well as being the most accurate method to determine left ventricular mass (an important clinical measurement in certain types of cardiomyopathy and heart failure), can also determine the cause of heart failure by quantifying microvascular perfusion, myocardial iron and fibrosis (Karamitsos et al., 2009). Diagnostic imaging of the myocardium using radio- or nanoparticlelabelled probes (‘molecular imaging’) is an area of active development (Saraste et al., 2009) and is potentially one of the quickest ways to translate advances in basic science into the clinical arena. Many of these techniques are still experimental in that they have not yet been proven to predict clinical events.

Case study

A 72-year-old man was admitted to hospital complaining of a progressive three-day history of shortness of breath at rest, exacerbated by lying flat. He had suffered a myocardial infarction four years earlier, which was treated by primary angioplasty. His past medical history was significant for hypertension, hypercholesterolaemia, and type II diabetes, which was diagnosed at the time of his infarct. Following the angioplasty, he was prescribed a β blocker, an ACE inhibitor, a statin, anti-platelet agents (all of which have been shown to reduce mortality following myocardial infarction) and oral anti-diabetic medication. He had remained well in the intervening four years. On examination, he was found short of breath at rest with crackles audible in both lung fields, consistent with left ventricular failure. Echocardiography was performed, revealing a significantly depressed left ventricular ejection fraction and normal valvular function. He was treated with intravenous diuretics to good symptomatic effect, and had the doses of his other cardiac medications increased. Over the next six months, he had two further admissions with left ventricular failure, both of which were treated with diuretics.

The gene expression changes that lead to heart failure are not definitively known. Many changes in gene expression have been described in biopsies taken from human heart failure patients. However, biopsies are necessarily small and usually taken blindly through the endocardial route via a vascular catheter. The logistical, ethical and legal difficulties in obtaining high-quality biopsies from relevant patient groups have been addressed by a working group of the American Heart Association and European Society of Cardiology (Cooper et al., 2007). In the absence of widely available biopsy material, clinical trials and medical practice have relied on the use of biomarkers that are essentially unvalidated. A comparison of the gene expression changes in animal models and human failing hearts is vital for validating the findings from animal models of heart failure, and eventually for assaying biomarkers used in humans.

Table 1.

Commonest documented causes of heart failure

Unanswered clinical questions in heart failure

It is largely unknown whether the individual changes in gene expression and physiology that are observed in heart failure patients are adaptive or maladaptive, and how this changes with the evolution of the disease. Further questions include whether there are novel biomarkers (Lainscak and Anker, 2009), and which imaging modalities are optimal that will facilitate clinical decision-making in heart failure patients.

Two obvious deficiencies are hindering the development of new heart failure therapies: (1) high-resolution, longitudinal phenotyping of heart failure patients (i.e. at several stages in the evolution of the condition) has not yet been carried out; and (2) the development of new animal models that more closely mimic the medical treatment of heart failure, and the common causes of heart failure (such as obesity and hypertension) that interact with treatment, would facilitate convergence between clinical and animal modelling fields. Given the huge recent advances in our understanding of murine biology, mouse models would arguably be of the most use to answer these questions.

Recent insights into cardiac biology

The last two decades have seen rapid advances in our understanding of the genetic and physiological processes involved in the development and maintenance of a healthy mammalian heart, and how perturbations may result in disease; for example, the discovery of autologous cardiac repair through pluripotent cells, which has been extensively reviewed elsewhere (Chien et al., 2008Hansson et al., 2009Nakano et al., 2008). Development of this technology towards the clinic requires reliable animal models, which arguably do not exist yet.

Strikingly, the failing adult heart resembles the foetal heart in many ways. Several gene expression changes that have been reported in failing hearts are consistent with ‘re-expression of the foetal gene expression program’. The expression of numerous transcription factors that are associated with heart development (Rajabi et al., 2007), gene activation programmes via histone deacetylases (HDACs) (Molkentin et al., 1998), and foetal-type microRNA (miRNA) expression profiles (Rooij et al., 2006Thum et al., 2007) have been reported in failing hearts. Furthermore, ‘foetal’ contractile protein isoforms and ion channels have been documented in human failing hearts. An important question is whether this gene expression shift contributes to the heart failure phenotype, or whether it is a protective response. Recently, it has been postulated that foetal isoforms of the contractile protein myosin are beneficial in the failing heart as they reduce oxygen consumption, albeit at the expense of reduced contractile function (Krenz and Robbins, 2004Lowes et al., 1997Rajabi et al., 2007Tardiff, 2006). It is perhaps a paradox that reducing cardiac contraction in a condition defined by an insufficiency in cardiac output should be beneficial. However, there is supporting evidence from human adult therapeutics for this hypothesis. When administered appropriately to heart failure patients, β