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The advent of modern mouse genetics has benefited many fields of diseased-based research over the past 20 years, none perhaps more profoundly than cardiac biology. Indeed, the heart is now arguably one of the easiest tissues to genetically manipulate given the availability of an ever-growing tool chest of molecular reagents/promoters and “facilitator” mouse lines. It is now possible to modify the expression of essentially any gene or partial gene-product in the mouse heart at any time, either gain- or loss-of-function. This review is designed as a handbook for the non-mouse geneticist and/or junior investigator to permit the successful manipulation of any gene or RNA product in the heart, while avoiding artifacts. Here guidelines, pitfalls, and limitations are presented so that rigorous and appropriate examination of cardiac genotype-phenotype relationships can be performed. This review utilizes examples from the field to illustrate the vast spectrum of experimental and design details that need to be considered when using genetically modified mouse models to study cardiac biology.
Genetic engineering in the mouse has revolutionized many fields of disease-based research because it permits the isolation of a single gene to examine its function within the context of an integrated physiological system that shares the mammalia class with humans. In fact, the tremendous impact that gene-targeting technology has had on modern investigative disease-based research was recognized by awarding the Nobel Prize in Medicine in 2007. Prior to modern genetic engineering in the mouse only lower phylogenic organisms, such as Caenorhabditis elegans, Drosophila melanogaster, and Xenopus laevis permitted routine interrogation of single gene function from a systems biological and developmental standpoint, although these organisms are particularly inadequate for addressing more complex physiologic processes that often underlie heart disease in humans. Apart from model organisms, interrogation of single gene function also heavily relied on cultured cells grown in 2-dimensions in artificial medias with artificial substrates under highly contrived conditions, which obviously lacked physiological integration with other systems (tissues, cells, etc) and the correct neurohumoral milieu.
Cardiac hypertrophy and end-stage heart failure are whole organ phenomena that are impacted by mechanical stress and strain and occur within a complex neuroendocrine environment, a backdrop that cannot be adequately modeled in cultured myocytes or even in lower model organisms. In addition, genetic manipulation of disease-gene relationships in the mouse heart is now routinely possible given the characterization of reliable and tissue-specific promoters, as well as a large cadre of mouse lines that permit heart-specific genetic recombination at essentially any desired time point to either inactivate a gene or RNA species, or augment the function of a gene/RNA product. Moreover, the heart is a fairly uniform tissue with a restricted number of cell types that is functionally dominated by the cardiac myocyte itself, making it easy to manipulate genotype-phenotype relationships through myocyte-specific promoters. Cardiac myocytes have a relative lack of cellular turnover or proliferation and they are highly differentiated, collective features that permit stable genetic manipulation. Thus, the use of genetically modified mouse models has become a mainstay for cardiac biology in general, as well as for critically dissecting disease-gene relationships within the context of a functioning mammalian heart. However, the experimental design and technical procedures used in engineering the mouse heart are not trivial and if improperly implemented, can yield conflicting and spurious results. Here we have aggregated information on transgenic approaches to highlight the pitfalls and hazards intrinsic to using genetically modified mice. Guidelines are presented for properly conducting genetic manipulations of genes in the mouse heart so that reliable data sets are generated that more soundly predict disease-gene relationships.
Two fundamentally distinct gene-driven technologies, insertional transgenesis (transgenic mice) and gene targeting (knock-out (KO) mice), are used to permanently modify the mouse genome. Transgenesis defines the technical process of introducing an exogenous DNA element (transgene) by pronuclear injection of a newly fertilized oocyte1–3. The transgene to be injected can cover a broad range of DNA elements including but not limited to the following: a full or truncated cDNA, mutated cDNA sequence, a genomic DNA fragment, or an antisense sequence or a microRNA encoding element. These DNA regions are typically linked to a promoter to drive tissue-specific expression. As a technical note, it is critical that the bacterial-derived sequence used to propagate the plasmid DNA in E. coli be excised from the desired injection fragment, otherwise these bacterial sequences can inactivate the transgene by methylation and chromatin condensation. With insertional transgenesis, the transgene randomly integrates primarily within one site of the genome as either a single copy or more commonly, as a random number of copies that concatemerize4 (2 copies to upwards of 50 copies are possible). If the α-myosin heavy chain (α-MHC) promoter is used, the greater the number of inserted copies the greater the level of expression that is observed5.
By contrast, gene targeting harnesses an embryonic stem (ES) cell’s natural propensity for homologous recombination, hijacked to replace an endogenous gene using a properly designed targeting vector. The basics of gene targeting involve the use of a DNA targeting vector (constructed within a plasmid) that contains a selectable marker and 2 regions of DNA sequence homology designed to flank an area of genomic sequence that will be deleted once homologous recombination occurs in ES cells. The targeting construct is hence designed so that if homologous recombination occurs properly, the endogenous gene will be replaced with the targeting construct and the selectable maker in one of the 2 alleles, typically also causing deletion of 1 to several exons of the gene of interest. This technology can also be used to insert a mutation (knock-in (KI)) or insert another cDNA or exon region. The most vital steps in creating a targeted KO or KI mouse are the design of the targeting vector and strategy for genotyping. Careful preplanning at this stage can save an investigator time and resources. Because there are so many considerations when creating a gene-targeted mouse we have included a set of detailed guidelines for developing a knockout mouse in a supplement (Supplemental Information).
There are resources such as the knockout mouse project (KOMP, http://www.komp.org/) or the international gene trap consortium (IGTC, http://www.genetrap.org) that have repositories of targeted mouse ES cells or gene trap ES cells available for public use. The international knockout mouse consortium (http://www.knockoutmouse.org/) also provides an excellent database of all of the available targeting vectors, confirmed targeted ES cells, and gene trap ES cells available for a given gene. Investigators should be cautious when using gene trap ES cells because these mutant alleles are not precisely targeted and can sometimes result in ES cells positive for the selection marker but with a gene that is either not affected whatsoever or is hypomorphic in expression6–8.
The number one consideration for choosing which genetic technology to use when making a mouse model is the experimental goal itself. In transgenesis, the transgene’s expression is typically superimposed over the endogenous gene’s expression so this approach is traditionally used as a gain-of-function approach. However, transgenesis can also be used as a loss-of-function approach when the expressed cDNA encodes a dominant negative (dn) mutant protein, a partial protein, an anti-sense RNA, microRNA or shRNA. For example, an inhibitory transgenic strategy was effectively used in the heart to investigate the requirement of calcineurin (calcium-activated protein phosphatase that underlies cardiac hypertrophy) in pathologic cardiac growth. In these studies calcineurin function was inhibited through the overexpression of naturally occurring inhibitory proteins Cain/Cabin-1 (the calcineurin inhibitory domain)9, AKAP799, or RCAN110, 11 or through the overexpression of a dn-calcineurin12. The phenotypes observed in these calcineurin inhibitory transgenic lines, which showed inhibition of pathologic cardiac hypertrophy, closely matched the antihypertrophic phenotype of calcineurin Aβ (Ppp3cb) gene-deleted mice, which were designed later. This comparison suggests that the transgenic approach, despite involving gross overexpression, was effective at uncovering the phenotype of calcineurin loss-of-function in the heart13. Indeed, the more traditional usage of cardiac transgenesis, in which an activated mutant form of calcineurin was overexpressed in the heart with the α-MHC promoter, showed massive cardiac hypertrophy14.
Gene targeting is most often used to create deleted alleles for loss-of-function experimental strategies; however, KI models can be made in which a mutant version or selection cassette replaces the endogenous gene that in some instances can produce a gain-of-function like effect15. Replacement by gene targeting also employs the endogenous regulatory elements of the targeted loci, so that if a KI mutation is used, expression occurs at physiologic levels. For example, a gene-targeted KI strategy was used to create a mouse model of inherited hypertrophic cardiomyopathy (HCM) by replacing a single amino acid in the endogenous α–MHC gene locus, R403Q16. This mouse was instrumental in better understanding the molecular-genetic basis for the HCM clinical phenotype17–19. By comparison, Leinwand and colleagues generated a transgenic mouse model of HCM by overexpressing a mutant αMHC protein in the heart using a rat α-MHC promoter20, which revealed a disease phenotype consistent with the R403Q mutant KI mouse16, 21. These results again suggest that transgenesis can be just as informative as gene-targeting if properly designed, and typically many times faster with considerably less cost. Despite these considerations, the gene-targeting based approach (such as a KI mutation) is often more elegant and can be more physiologic.
Gene targeting technology can also be used to “knock-in” a reporter gene (i.e. β-galactosidase) into a locus of interest to identify temporal and spatial patterns of gene expression during cardiac development or in response to stress. For instance, to study the role and expression of islet1 (Isl1) in murine heart development a nuclear β-galactosidase construct was “knocked-into” the isl1 locus22. This mouse was then used to visualize myocyte lineage subdomains that express β-galactosidase as a surrogate for Isl1 during development.
While both transgenesis and gene-targeting are powerful experimental approaches, there are pitfalls associated with each that should factor into an investigator’s decision-making. These pitfalls are listed in Table 1 and more thoroughly reviewed below (Table 1). Perhaps the main drawback of choosing gene targeting is the considerable amount of time and money it takes to generate the mouse, assuming that one will be successful in targeting the desired locus in the first place (not a guarantee). Another issue with gene targeting is that many genes play vital roles during embryonic or early postnatal development, so that traditional somatic targeting in the mouse (absent from zygote stage onwards) might produce lethality, precluding an analysis of an adult heart disease-related phenotype. Indeed, we were unable to ascertain the adult cardiac hypertrophy-associated phenotype of ERK1/2 in the heart using traditional gene-targeting because Erk2 null mice die early in embryonic development23. We later bypassed this developmental lethality issue using Cre-LoxP technology to generate heart-specific deletion of Erk2 in the Erk1−/− background24. In addition to embryonic lethality, another problem is that the adult cardiac phenotype observed in a traditionally targeted KO mouse could be influenced by loss of gene function in other organs or tissues. Finally, replacement of the endogenous gene at a given locus can alter transcriptional patterns of neighboring genes due to the deletion of their critical regulatory elements even with a precise targeting construct or from interference with the neomycin cassette and its promoter. Knowing as much as possible about the genomic locus of interest through comparative genomic programs can guard against this situation and is further discussed in the supplement (Supplemental Information). A classic example of this was identified when three different labs independently deleted the muscle regulatory factor MRF4. Interestingly, 2 of the 3 MRF4 KO models were lethal during development while the other was not. In the 2 non-viable models, expression from the neighboring Myf5 gene was also absent despite the Myf5 locus being intact25–27.
Transgenesis also has various technical challenges due to the fact that the transgene randomly inserts into the genome and that expression levels are typically higher and can be considered “non-physiologic” (Table 1). However, we have generated transgenic lines where expression almost exactly approximates the known increase in expression of a disease-associated gene in the heart. For example, thrombospondin 4 is induced in the heart following many different disease-inducing stimuli, and this level of induction was approximated by transgenesis with the α–MHC promoter, accurately revealing its disease modifying phenotype28.
Another issue that can seriously compromise a genetic based experiment in the mouse is if more than one gene or transgene to be simultaneously manipulated resides on the same chromosome. The easiest way to understand the issue is by example from our own laboratory. We were attempting to develop double null mice between the δ-sarcoglycan (Sgcd) and the JNK2 (Mapk9) deleted loci to determine if loss of JNK2 protein might impact muscular dystrophy pathogenesis in mice lacking δ-sarcoglycan. We crossed the mice, but their offspring were never genotyped as double homozygous. Only double heterozygotes were ever identified. Checking the genomic database we easily surmised that the reason was because both genes reside on mouse chromosome 11. This same issue can befall a transgene if one is trying to cross it into a homozygous null gene-deleted background. Moreover, transgenes are rarely mapped for chromosomal insertion, and when one fails to identify homozygous nulls with the experimental transgene of interest, most of us would interpret it as a lethal event due to the biology associated with the cross, when actually it is because transgene and targeted allele are on the same chromosome. However, if the genes/transgenes of interest are far enough apart on the same chromosome, meiotic cross-overs can occur with enough breeding, which will allow for success in obtaining double nulls or a null loci with a hemizygous transgene. Finally, if maintained in the hemizygous state, 2 transgenes are easily crossed even if they reside on the same chromosome, but neither can be made homozygous without obtaining cross-overs (and if the loci of interest are too close together on one of the chromosomes, such events can be extremely rare).
Assuming that transgenesis is the technology of choice for engineering one’s mouse, the next step is the selection of the appropriate promoter to govern your transgene’s expression. Choosing the right promoter is a vital component of your construct’s design as it determines the level, cellular and tissue specificity, as well as temporal-spatial pattern of transgene expression. While the overall design of a construct is relatively simple and one’s choice of promoter would seemingly rule the day in directing expression as desired, the transgene’s expression can be influenced by regulatory elements within the random locus of integration. There are a number of promoters listed in Table 2 and schematically illustrated in Figure 1 that can be used to gain robust expression of a transgene in cardiac muscle (Fig 1A). Some important considerations when choosing a promoter are (1) in what cell type is the promoter active, (2) where in the heart or other tissues does it express, and (3) when does the promoter drive expression-developmentally or in the adult heart. As illustrated in Table 2 each promoter turns transgene expression on or off at different developmental time points and in different regions of the heart or other tissues, which needs to be considered when selecting the correct promoter as well as when analyzing the phenotype of the mouse.
While this review will not detail all of the cardiac promoters in Table 2, it will cover the most highly used promoters for expressing a transgene in the postnatal/adult myocardium. The α–MHC promoter provides robust expression in ventricular myocytes that “appears” just a few days after birth (Table 2). However, this promoter is also transiently active for a brief window during primitive heart tube development and for all of development in the atrial region29, 30, thus developmental problems could still arise due to this transient early expression. The α–MHC promoter is perhaps the most commonly employed of the cardiac promoters because it is highly specific to the cardiac myocyte without “leaky” expression in other cell types. Even the so-called “ectopic” expression from this promoter observed in limited regions of the lung occurs within cardiac myocytes of the pulmonary myocardium31. A 5.5 kb region of the α–MHC locus, which contains the entire promoter region, was developed by Jeffrey Robbins and James Gulick and has become the mainstay construct in our field5. Importantly, this 5.5 kb region contains uncharacterized insulator elements that protect it from the influence of the surrounding locus in which the transgene integrates, so ectopic expression is rarely observed, and almost every line shows good expression in the heart. These insulator elements also produce a rather linear relationship with copy number-dependent expression level (more copies integrated gives higher expression levels5). The robustness of insulation in this construct also means that only 2–4 founder lines are needed to obtain at least 2 satisfactory lines for full analysis. A minor drawback to the α–MHC promoter is that it is downregulated by disease stimuli that induce hypertrophy or during heart failure (β-MHC promoter activity is induced)32, 33, or if thyroid hormone levels drop5, necessitating re-evaluation of one’s data if these disease states are also part of the phenotype because expression can decrease as the disease evolves.
There are several other promoters listed in Table 2 that have reasonable cardiac muscle specificity and have been used for transgenesis, such as the β-MHC promoter34, 35, the ventricular myosin light chain 2 (MLC2v) promoter36–39, and cardiac troponin T (cTnT) promoter40, 41. While useful, these three other promoters often show lower transcriptional activity in the adult heart compared to the α–MHC promoter, and more importantly, these 3 other promoters are not as well insulated so several lines need to be carefully examined to ensure expression in the heart without significant expression in other tissues. An additional consideration with both the MLC2v and β-MHC promoters is that they drive expression in slow skeletal muscle. Moreover, all 3 of these alternate promoters have robust developmental expression in the early heart tube, which is often why they are used in the first place for studying cardiac development. With respect to insulation, while ectopic expression outside of the heart could make a given line of less utility, sometimes low-level transgene expression in the heart, as driven by these 3 alternate promoters, may be desirable. For instance many signaling proteins are better analyzed at lower levels of expression to guard against loss of endogenous regulation. In fact most physiologic increases in gene expression are only several fold above endogenous levels. As a case in point for how expression levels are a serious consideration in interpreting results of a transgenic experiment one merely needs to consider work from the Dorn laboratory. Overexpression of an inhibitor peptide (δV1) of the δ isoform of protein kinase C (PKC) with the standard α–MHC promoter caused early morbidity due to heart failure42. To circumvent this early lethality and obtain viable δV1 mice to study, the authors created an attenuated α–MHC promoter that retained cardiac muscle specificity but dramatically reduced expression levels (Table 2). This was achieved by ablating either the first or second thyroid response element (TRE) in the α–MHC promoter. Hence, one would have initially interpreted that inhibition of PKCδ was pathologic to the heart (in contrast to prkcd null mice that are viable without a major cardiac baseline phenotype43–45), but if the attenuated promoter was used one would have interpreted the opposite, because lower levels of overexpression of the δV1 inhibitory peptide protected from cardiac ischemia-reperfusion injury42.
In other areas of cardiac biological investigation, high levels of transcript overexpression are absolutely required to achieve the desire effect, such as when studying sarcomeric proteins and the effects of mutations. For such experiments the amount of transgenic transcript must outcompete or dilute the endogenous transcript so that the desired myofilament protein is effectively replaced46. In this case the total contractile protein quantity remains unchanged despite significantly increasing total transcript expression, because the stoichiometry of the sarcomere is maintained46, 47. The α–MHC promoter has been used extensively for studying sarcomeric proteins because transgenic mice can be obtained with a range of replacement levels that can approach nearly 100%.
An alternative to the traditional transgene design in which a gene or cDNA of interest is linked to an exogenous cardiac specific promoter is to employ artificial chromosome vectors to direct transgene expression. The 3 categories of these vectors are yeast (YAC), phage (PAC), and bacterial (BAC) artificial chromosomes that are designed to accommodate large genomic inserts of 100–300 kb or larger in the case of YACs48. Artificial chromosome vectors can be used for the same applications as a traditional transgene, and they integrate into the genome randomly like a transgene (usually just 1 copy), but their real experimental power is producing expression patterns and levels that highly mimic the endogenous gene49. These artificial chromosomes can contain the entire gene of interest in its natural intron-exon arrangement with its “full” complement of regulatory elements, or one can insert a cDNA into a gene locus within the artificial chromosome in the hopes of obtaining expression of that cDNA in a pattern and level that exactly mimics the locus into which it was placed.
For some experiments it is highly desirable to induce transgene expression for the first time in the adult heart, hence bypassing early postnatal developmental expression, or to turn off expression in the adult heart once disease has been induced. Such control is critical for discerning the acute verses compensatory effects of a genetic manipulation in vivo as well as the disease-specific effects of a given gene’s function. There are several inducible transgene systems to regulate expression in a temporally restrictive manner, but the most effective strategy is based on the tetracycline (Tet) regulatory system. Here controlled induction of your gene of interest requires two transgenes50–52: (1) a Tet transactivator (tTA) cDNA under the control of a tissue specific promoter (i.e. α–MHC) and (2) a conditional transgene that is cloned downstream of a minimal cytomegalovirus (CMV) promoter coupled to multiple copies of the Tet-operator DNA elements (Tet-O). The Tet transactivator functions as a transcription factor that initiates expression of any gene downstream of its putative promoter element, the Tet-operator. In the “Tet-off” system tetracycline or its analog doxycycline are given to block the interaction between tTA and the Tet-O, which in turn silences the transcription of your transgene. The “Tet-on” system utilizes a mutant tTA to essentially cause the reverse of the Tet-off system such that doxycycline is required to activate transgene transcription50–52.
The conventional Tet-off system based on the CMV promoter for inducible transgenesis in the heart was not successful and only produced low levels of unregulated expression52. However, the Tet-Off system was re-engineered for the heart by Jeff Robbins’ laboratory such that the 5.5 kb mouse α–MHC promoter was modified to contain the Tet-O, along with deletion of key transcriptional enhancer binding sites to attenuate the greater promoter52. The Robbins laboratory also generated a very low expressing tTA mouse line that was non-toxic using the “standard” 5.5 kb mouse α–MHC promoter (Fig. 1B)52. Others have shown that slightly higher levels of tTA, such as with the rat 2.9 kb α–MHC promoter, induced hypertrophy and cardiomyopathy on its own53. The reengineered system by the Robbins laboratory is non-leaky, fully inducible, and completely cardiac-specific52. Another benefit of this inducible system is that the α–MHC-Tet-O promoter construct can be used singularly, without the α–MHC-tTA transgene, as an attenuated promoter that gives lower and more “physiologic” levels of overexpression for baseline analysis. However, cDNAs driven from the attenuated α–MHC-Tet-O promoter construct still afford the opportunity for inducible expression at a later time if one then crosses in the α–MHC-tTA transgene (unpublished observations). As a final consideration, Sanbe et al52 demonstrated that 6 months of chronic tTA expression in mice is innocuous, thus studies using the Tet-O system should probably be completed within 6 months to avoid any complications from tTA toxicity, although a control group of α–MHC-tTA only transgenic mice should always be used, especially if experiments are planned past 6 months. An example of using an adult-specific inducible transgenic approach was recently published by Joseph Hill and colleagues, where the ability of calcineurin to induce hypertrophy in the adult mouse heart was examined54. It was previously shown that activated calcineurin driven by the standard α–MHC promoter induced massive cardiac hypertrophy14, but this hypertrophy was present by 1 week after birth so that developmental effects could have influenced the continued growth of the adult heart. However, use of a tTA-dependent system in which the activated calcineurin cDNA was induced for the first time in the adult heart, validated the ability of this signaling pathway to mediate adult cardiac hypertrophy without a developmental complication54.
Another means of achieving inducible expression of a cDNA in the heart involves using loxP-stop-inactivation cassettes contained within a traditional transgene, so that the Cre-loxP system can be used to temporally regulate a transgene (discussed more under gene-targeting) (Fig. 1C). A complete understanding of this system is best described with the following example. Yibin Wang and colleagues generated an α–MHC promoter construct that contained an upstream green fluorescent protein (GFP)-loxP flanked cassette, followed by the cDNA of interest. They selected an activated mitogen-activated protein kinase kinase 7 (MKK7) mutant protein to force c-Jun N-terminal kinase (JNK) activation in the heart, after Cre-mediated excision of the GFP cassette, which revealed a role for JNK as a mediator of cardiomyopathy55. This system is beneficial as it allows generation of founder lines that are inactivated and hence not subjected to lethality if the cDNA if interest causes such. The recombination of the loxP-dependent cassette in the transgene is permanent once it is crossed with a Cre-expressing line, after which expression persists for the cDNA of interest. When used with the αMHC-MerCreMer, tamoxifen inducible line (Fig. 1D), one can achieve temporally regulated and cardiac-specific gene activation in the heart56. A similar strategy can also be employed using any ubiquitous promoter that has the loxP-dependent stop cassette, but only when used in conjunction with a cardiac-specific Cre. For example, Yutzey and colleagues used a CAG-CAT ubiquitous promoter (CMV enhancer-fused to chicken β-actin promoter) with a chloramphenicol acetyl transferase (CAT)-loxP dependent stop cassette to permit conditional transgene expression in the developing heart of a downstream Tbx20 cDNA, when crossed with β–MHC-Cre transgenic mice57 (Fig. 1C). The benefit of the CAG-CAT transgene is that one is not limited to the heart, as any tissue where Cre is expressed can be subjected to tissue specific gene/cDNA induction, and if a drug inducible Cre line is used, recombination of the recipient loxP-dependent transgene can be temporally controlled as well.
One pitfall to navigate is the exclusive use of the founder transgenic line for phenotypic analysis. The founding mouse (F0) for a given line should only be used for breeding due to the potential for mosaicism. All transgenic founder mice should be backcrossed with wildtype mice of their same native strain (typically C57BL/6 or FVBN) to propagate the transgene and obtain germline transmission (F1 generation). Founder transgenic lines (F0) are the direct result of the pronuclear injection of newly fertilized oocyte, which can generate mosaicism if the DNA injection fragment integrates into the genome after the first zygote cell division to the 2 or even 4 cell stage, meaning that only half or a quarter of the cells in the generated founder mouse contains the transgene. F1 progeny from these founders, when positive for the transgene, are now germline and hence all cells are positive for the transgene. These germline mice should then be used for all subsequent breeding and experiments (Table 1).
Another issue to consider is the number of copies that insert, and the number of potential independent integrations that occur in a given line (Table 1). Conventional thinking might predict that a transgene integrates into the mouse genome at one site, as a single copy event. While it is usually true that only a single integration site is observed per founder line, most often this event, as discussed earlier, contains multiple concatemeric copies of the transgene (5 to 20 copies seems about typical). Given this characteristic, one can easily mix 2 different transgene fragments together prior to oocyte injection to generate matched expression levels between 2 proteins. For example, this technique permitted co-expression of dn cDNA versions of the stress induced signaling proteins JNK1 and JNK2 in the heart at roughly the same levels from a single insertion site58.
On rare occasions more than one integration site can occur in a single founder mouse (meaning that a single founder mouse (F0) can give more than 1 line, Table 1)59. For example, we generated transgenic mice expressing constitutively active MKK1 in the mouse heart under the control of the α–MHC promoter60, and one of the F0 mice gave litters that were nearly 100% transgenic, when the predicted Mendelian frequency should be 50% from a single integration site when bred with a nontransgenic mouse. When this MKK1 F0 mouse was bred into the second (F1) and third (F2) generations we were able to account for 3 separate genomic integrations on 3 different chromosomes, each with its own unique expression level and each representing an independent line (unpublished observations).
Another issue to consider is whether to keep the transgene hemizygous or to interbreed the same line onto itself with the hopes of obtaining homozygous mice to increase expression levels. However, we discourage attempting to generate and keeping transgenic mice as homozygotes for 2 reasons. First, genotyping is often difficult to discern between 1 versus 2 dosages of the insertion, and guessing from a PCR reaction for a presumed 2-fold increase in signal is never reliable. The only proven way of determining a hemizygote versus a homozygote for a given transgene insertion is to breed each F2 animal back to a wildtype, and if the mouse in question was hemizygous, 50% of the progeny will be transgene positive, but if it was homozygous, 100% of the progeny will be positive as hemizygotes. However, the bigger concern with homozygosity of a transgene is that it can affect the function of the gene in which the transgene integrates, as it is not uncommon for a transgene to disrupt or alter expression of the gene in which it inserts, which is typically not a problem in the hemizygous state (the other allele is still intact). If it becomes necessary to identify the transgene’s integration site, there are PCR-based strategies for determining its chromosomal location in which genomic sequences flanking the transgene are consecutively amplified (inverse PCR and genome walking61, 62 and then these fragments are sequenced and referenced against the mouse genome.
Another issue to consider in cardiac transgenesis is the loss of expression over time due to loci inactivation associated with methylation (Table 1). The actual chromosomal location of the transgene can play a role in determining the level and pattern of transgene expression, and if the transgene integrates into a transcriptionally inactive or silenced genomic region it may be more susceptible to silencing due to methylation and irreversible chromatin condensation63. Not all transgene constructs have the same susceptibility to this transcriptional silencing, and the α-MHC promoter is probably the least likely to be silenced over time given its strong insulation. The nature of the cDNA insert can also have a dramatic effect, especially if it is prokaryotic or from certain species that have high GC content in their transcripts63, 64. The best rule of thumb is to use a mouse cDNA with a mouse-derived promoter, when possible, to guard against transgene silencing. A related issue is that once a transgene silences in a given mouse within one’s colony, all its progeny will have the transgene silenced, but the exact same transgenic line could still express in other parallel or older mice from one’s colony. Hence, one should never maintain a transgenic line with only one breeder, and expression should be periodically verified when maintaining a line long-term.
The initial phenotype one identifies with a newly generated transgenic line can change over time. As a line is continuously bred within one’s colony, modifying mutations arise over the years that give a selective advantage towards reducing a disease phenotype. For example, we originally generated transgenic mice expressing activated calcineurin in the heart over 14 years ago, which induced massive HCM that was lethal within 3–5 months14. However, some 14 years later mice from this same line now routinely live well over 1.5 years in our colony, despite strong overexpression as originally characterized with massive cardiac hypertrophy (unpublished observations). To prevent issues with genetic drift the Jackson Laboratory recommends refreshing breeders on a regular basis and after 5 generations a new foundation stock should be developed from cryopreserved pedigreed embryos.
A transgene can also insert into a sex chromosome, which can be problematic for 2 reasons (Table 1). First, if the transgene integrates into the X chromosome it can be subjected to X-linked inactivation in females, leading to mosaicism in expression. Also, transgenic males from such a line will never give rise to transgenic males when bred with wildtype females, so only females can be analyzed from such an intercross. The second concern is if the transgene integrates into the Y chromosome, because it will never be possible to assess the effect of the transgene in females. Thus, it is generally a good rule to avoid any lines in which the transgene is incorporated on either the X or Y chromosomes.
Another issue to consider is that essentially all standard (non-inducible) promoters used for generating a cardiac-restricted transgene have some or a great deal of expression in the developing mouse heart (Table 2), which if the lines survive into adulthood, could still impact the heart due to a prior developmental effect. For example, Dorn and colleagues showed that overexpression of the heterotrimeric protein Gαq in the heart with the “standard” α–MHC promoter caused disease in adulthood characterized by hypertrophy and myocyte dropout65. However, this same Gαq gene protein product, when overexpressed at the same levels but for the first time in the young adult mouse heart using an inducible system, produced no disease, suggesting that the early developmental or postnatal expression of Gαq was the responsible factor in producing or sensitizing to adult disease66. Hence, it may be desirable to use the inducible α–MHC-Tet-O system as a means of isolating the function of a gene product to the adult heart. However, as described earlier, use of this inducible system to overexpress activated calcineurin in the heart, in contrast to results observed with Gαq, did still show that disease could be driven exclusive to the adult heart without secondary influences from earlier developmental expression66.
Another issue to consider when using any of the α–MHC-based promoters is that of atrial enlargement due to myocyte hypertrophy, proliferation, fibrosis and thrombi formation. The α–MHC promoter is continuously expressed in the atria of the heart throughout all of embryonic development, as well as at much higher levels in the atria of the postnatal and adult heart compared with the ventricles. Thus, even wildtype control proteins, such as the myosin light chains (MLC), when overexpressed at high enough levels, can lead to atrial disease that could eventually impact the ventricles67. Thus, we often avoid any sort of atrial analysis in our transgenic experiments, as they can show artifactual results just based on the extreme level of persistent overexpression in this region of the heart. Thus, if one intends to study atrial biology or SA node function in the heart, the α–MHC promoter may not be the best choice. As an aside, we have noted that the α–MHC promoter drives expression in the SA and AV nodes, as well as the rest of the conducting system of the heart (unpublished observations).
There are a number of additional factors listed in Table 1 that can complicate the phenotypic interpretation of transgenic experiments. Some of these factors include: (1) “Molecular torture” in which high levels of protein expression cause a nonspecific phenotype or aggregation of a protein of interest; (2) Protein toxicity effects in which epitope tags or reporter constructs (ie. eGFP) cause non-specific phenotypes or lethality; and (3) Poison peptide or partial protein effects in which aberrant splicing events in the transgene generates dominant negatives that give an unanticipated phenotype, or again, protein aggregation. One can circumvent these complications with carefully designed experiments such that the total level of overexpression is carefully controlled, appropriate controls are used to rule out effects of epitope tags or bacterial protein fusions, or if aberrant splicing is observed, one can select a new cDNA from a related species for overexpression (Table 1 and Figure 2). In addition the Jackson Laboratory through the mouse genome informatics website (http://firstname.lastname@example.org) offers an online forum that discusses a wide range of topics that can be extremely helpful for designing experiments and circumventing these pitfalls.
Another good practice is to always analyze more than 1 transgenic line, and more importantly, to analyze various levels of overexpression across at least 3 independent lines. For example, the role of the calcium regulatory protein phospholamban (PLN) was carefully examined by transgenesis. For each experiment nontransgenic and transgenic littermates from different lines with various degrees of overexpression were studied in order to develop a gene dose-response relationship68. One should also routinely determine if the protein that is overexpressed is still properly localized to the desired intracellular compartment, that it is not degraded and absent (this sometimes happens with truncated proteins or peptides), that a signaling or regulatory protein of interest gives more activity in higher expressing lines, and that expression is maintained over time at the expected levels in the ventricles.
While gene targeting (KOs and KIs) is considered a more precise strategy for manipulating the murine genome when compared to insertional transgenesis, there are a number of theoretical and technical pitfalls to consider (Table 1). It is always important to carefully assess other tissues that might be affected in total somatic KO mice, which could secondarily impact the heart. However, there can be exceptions to this concern. For example, deletion of the gene encoding phospholamban (Pln) was engineered with a traditional, global gene targeting strategy, and despite the lack of tissue specificity, its function in the heart was still readily characterized without secondary effects, despite its deletion from skeletal muscle and smooth muscle where it is also appreciably expressed69. Indeed, while slow skeletal muscles from Pln KO mice did demonstrate a significant change in contractile function70, it did not impact the cardiac phenotype, although this may not be the case for other genes that are deleted globally. Hence it is always important to carefully assess other tissues that might be affected in total somatic KO mice that could secondarily impact the heart. As will be discussed below, the only definitive means of establishing the myocyte autonomous function for a given gene through gene-targeting is to use a cardiac-specific approach
While gene targeting typically offers a definitive means of extinguishing gene function, the traditional strategy that results in the total somatic (global) gene deletion can be subject to secondary and compensatory effects that mires interpretation of cardiac function/effects. First, total somatic deletion of a gene can result in embryonic lethality due to its necessity, sometimes even for proper heart maturation and function. Second, total somatic deletion of a gene is more likely to uncover and induce compensatory changes in other genes as a means of bypassing lethality or altered homeostasis during development. Also, such putative compensatory changes in other genes might not normally occur under any other conditions, thus obscuring a proper assessment of one’s gene of interest. Third, as discussed above, total somatic deletion of a gene can alter the heart through secondary affects associated with loss of said gene in other organs or tissues. For example, deletion of the transcription factor Mef2c in the mouse produced embryos with poorly developed hearts, resulting in early lethality, at first presumed to be solely due to its loss in the myocytes of the heart71. However, it was later determined that global deletion of Mef2c also produced embryos without an organized vasculature, which can lead to embryonic death itself with a secondary defect in heart maturation72.
To circumvent these three main limitations plaguing total somatic gene targeting strategies in the mouse, tissue-specific approaches have become widely adopted by the research community. Cre-loxP technology has become the preferred means of generating tissue-specific gene deletion in the mouse. This approach utilizes the DNA site-specific Cre recombinase (Cre, 38 KDa), which is typically driven by a tissue-specific transgene or even an endogenous gene loci if Cre was inserted by gene-targeting. Once expressed, Cre recombinase recognizes a 34-bp DNA sequence element referred to as a loxP (fl) site, which is engineered into the desired gene loci for future manipulation. Placement of 2 loxP sites flanking exons of interest in a target gene permits subsequent deletion of those exons when Cre is introduced at a later time. Placement of the small loxP sites by traditional gene-targeting is typically innocuous to the selected locus, although it is good to verify that expression of the targeted locus was not altered by introduction of these small sequence elements (after the antibiotic selection cassette has been removed). Heterozygote loxP-targeted mice can also give an intermediate phenotype compared with homozygotes, and can be included in ones experimental group. However, often times heterozygosity of a loxP-targeted allele has no discernable effect. Hence, in our laboratory we rarely analyze heterozygote loxP-targeted mice for purposes of economy, unless we suspect that a gene-dosage effect is in play.
There are several Cre transgenic lines available for cardiac expression that are listed in Table 2. The temporal and spatial pattern of subsequent gene deletion is regulated by the promoter or endogenous locus that drives expression of Cre. For example the Nkx2.5-Cre KI mouse line expresses Cre in the developing heart beginning around day E7.573. By 1 day after birth nearly 100% excision of the loxP flanked DNA region has been reported with this KI line, making it one of the strongest alleles for Cre-mediated excision in the heart (Table 2). However, the Nkx2.5-Cre KI allele is not absolutely specific to cardiomyocytes, so deletion can occur in other sites of the embryo/adult mouse. Also to consider, the KI approach deletes one functional copy of the Nkx2.5 gene, which can itself render a phenotype as shown previously74, 75.
Other Cre-expressing lines are also available, though most are driven by a transgene with various promoter options that produce variable excision efficiency in the heart (Table 2). Both the α–MHC-Cre and β–MHC-Cre offer excellent excision efficiency in which 70–90% of gene expression is lost76. Using the β–MHC-Cre transgene gives strong DNA excision during early cardiac development (E7.5 onwards)76 and then ceases shortly after birth, thus limiting any potential toxicity associated with chronic Cre expression in the adult heart. As a word of caution, the β–MHC-Cre will also induce gene excision in slow skeletal muscle of the mouse so these effects will need to be considered. The β–MHC-Cre transgene will also be induced and re-expressed in the adult heart with disease stimulation, which could produce further levels of gene deletion that was not anticipated (the β–MHC gene is induced in the mouse heart by hypertrophic stimuli).
The α–MHC-Cre transgene can have transient expression during early embryonic development in the heart, so it could still produce developmental effects and even lethality depending on the gene that is being deleted. This transgene is then mostly shut-down in mid to late embryonic development of the ventricles (persists the entire time in the atria), only to be robustly induced in the postnatal ventricles of the heart where it is maintained at high levels into adulthood. However, this persistent expression of Cre recombinase in the adult heart is not without potential concerns.
For example, the widely used mouse Cre line developed by Schneider and colleagues, under control of the α–MHC promoter77 causes toxicity leading to cardiomyopathy by 8–12 months of age (unpublished observations). Hence, we attempted to develop our own α–MHC-Cre expressing transgenic line. Figure 3 illustrates several α–MHC-Cre lines with high, medium, and low expression of Cre protein in the heart (Fig. 3A). By 6 months of age both the highest (line 10.10) and lowest (line 12.10) expressing lines presented with reduced cardiac function and cardiomyopathy (Fig. 3B and 3C), with increased heart weight to body weight ratios (data not shown). The lower expressing line (12.10) might be cardiomyopathic because of a combined effect of transgene integration and Cre expression, especially since the other low/medium expressing Cre lines (10.6, 10.14, and 11.8) exhibited no change in cardiac growth or function (Fig. 3). These results underscore how transgenesis in general can lead to cardiac disease, in this case due to a toxic effect of Cre that is probably related to its ability to cause genomic instability and chromosomal rearrangements78. However, this initiative did result in the identification of a stable α-MHC-Cre line that we now routinely use76, which does not predispose to cardiomyopathy as characteristic of the α–MHC-Cre line developed by Schneider and colleagues (Table 2). Our results also underscore how important it is to have a Cre-only transgenic group as a control (Fig. 2). These control groups will aid in differentiating the affects of the Cre transgene verses the excised targeted DNA region. In fact studies may need to be terminated early so as to avoid Cre-dependent cardiotoxicity if the α–MHC-Cre generated by Schneider and colleagues is used77. Alternatively, Cre could be delivered using β–MHC-Cre transgenic line, which no longer shows appreciable expression in the adult heart, or other alternative Cre lines could be used (Table 2).
An adjunct to tissue-specific gene targeting is to employ one of the few drug inducible Cre expression lines based on the α–MHC promoter (Table 2, Fig. 1D). These systems not only limit Cre expression to the heart, hence maintaining tissue-selectivity, but also provide temporal control over gene excision. In drug inducible constructs, Cre recombinase is fused to a mutated version of the ligand binding domain of estrogen receptor (MER), the most efficient version of which has a double fusion that is termed MerCreMer79. The mutated version renders the ligand binding domain insensitive to the endogenous ligand estradiol but sensitive to a synthetic analog, tamoxifen, which is given to activate the Cre so it can induce recombination80. Sohal et al linked a cDNA encoding this MerCreMer fusion protein to the α–MHC promoter for temporally regulated gene deletion approaches in the heart56. When crossed with the ROSA26-Stop-β-galactosidase reporter line, this α–MHC-MerCreMer mouse line showed almost no leakiness in the adult heart at baseline, but efficient loxP-site dependent recombination with administration of 5–7 days of tamoxifen56. The optimal tamoxifen dose and route of delivery can vary depending on the characteristics of the genomic locus that is targeted with LoxP sites and the half-life of the protein encoded by the gene of interest. Most publications deliver tamoxifen at 20–80 mg/kg daily over 4–5 days via intraperitoneal (IP) injection. Tamoxifen food is also commercially available and can successfully induce recombination by feeding an estimate of 80–100 mg/kg daily dosage over 4–7 days. For example in mice that have both floxed SERCA2A alleles and transgenic expression of α-MHC-MerCreMer, SERCA mRNA was reduced to less than 5% of that in controls after 4 days of either daily tamoxifen injections (80 mg/kg) or food (80–100 mg/kg)81.
As a cautionary note, tamoxifen administration to MerCreMer transgenic mice alone can induce an acute reduction in cardiac function with some associated remodeling but this typically resolves within 7–14 days after cessation of treatment82, 83 (unpublished observations). Interestingly, MerCreMer negative littermates receiving tamoxifen were unaffected as were vehicle injected MerCreMer positive littermates suggesting that neither the MerCreMer transgene nor tamoxifen alone affected cardiac function but rather it was the interaction between the two that was detrimental. To navigate this issue, an oral route of tamoxifen administration was shown to be just as effective as an injection in promoting recombination81, 84, but without the transient cardiomyopathy (unpublished observations). Alternatively, one can give raloxifene, a weaker estrogen analog that typically does not produce as robust recombination as tamoxifen, but also does not appear to induce transient cardiomyopathy83. One could also simply wait 2–3 weeks after tamoxifen treatment before assessment of the cardiac phenotype is attempted, so that any issues related to the tamoxifen-MerCreMer combination have resolved. Clearly, a tamoxifen treated MerCreMer-only transgenic group needs to be employed as an important control in all such experiments utilizing this line (Fig 2). Finally there is an alternative α–MHC driven inducible Cre (CrePR1) that uses the progesterone receptor ligand-binding domain as the basis for the Cre fusion protein, which is induced with RU486 (Table 2). However, the α–MHC-CrePr1 showed unacceptably high baseline leakiness of recombination in the heart without RU48685, hence the field has not adopted it.
Finally, tetracycline/doxycyline-dependent Cre-based transgenic systems have also been defined for cardiac-specific recombination. One of these systems utilizes a CMV promoter Tet-O transgene to drive Cre expression in any potential tissue, but if used with an α–MHC-reverse tTA (rtTA) transgene, it produces heart-specific deletion when tetracycline is given86. The other system involves the troponin T promoter to drive rtTA in the heart with the same CMV-Tet-O-Cre responder line (Table 2)87. These 2 lines of rtTA mice give another means of driving Cre expression specifically in the heart at any desired time point with the addition of a tetracycline, especially if tamoxifen is determined to be detrimental when the α–MHC-MerCreMer system is used.
An elegant example of how conditional gene targeting can provide “definitive” answers to longstanding controversial issues in cardiac biology was recently published. Lee and colleagues addressed the issue of ongoing myocyte replenishment and regeneration within the heart using a genetic “pulse-chase” (fate-mapping) system. This system labels all adult myocytes in the heart at time 0 with the α–MHC-MerCreMer transgene and a ubiquitously expressed ZEG88 cassette (β-galactosidase/eGFP) in the ROSA26 locus89. Loss of properly recombined myocytes over time or after injury presumably reflects the rates of new myocyte generation in the heart from non-myocyte sources. The authors found that after myocardial infarction induced injury or pressure overload, the percentage of baseline labeled cardiac myocytes had significantly decreased indicating that new myocytes were formed from precursor cells. By contrast normal aging showed no reduction in recombination-marked cardiac myocytes, indicating a lack of contribution from stem/precusor cell populations. As a whole this study provided an elegant example of how modern genetic approaches in the mouse can provide definitive answers in controversial areas of cardiac biology.
Mouse strain can dramatically affect the resulting phenotype of a genetic manipulation (Table 1). Each transgenic and ES cell core has their preferred mouse strain. Most conventional transgenesis is performed in FVBN or C57BL/6 strains90 while most gene-targeting is performed in SV129 ES cells, which typically generates targeted mouse lines with a mix of SV129 and C57BL/6 backgrounds91, 92. These are important considerations because strain dependent differences in baseline cardiac function have been reported in several studies93–96. The effects of strain differences can add a considerable source of variability that could partially explain differences between transgenic mouse models with similar genetic manipulations. For example, an E180G tropomyosin replacement transgenic mouse made on the C57BL/6 background exhibited no cardiac remodeling and only mild diastolic dysfunction97. By contrast the E180G tropomyosin mutation within the FVBN background resulted in massive cardiac hypertrophy and heart failure98. Additional strain dependent differences have been identified when measuring responses to ischemia reperfusion93, β-blockade93, and pressure overload99. Our lab has observed that FVBN mice are generally resistant to pressure-overload induced heart failure, even past 16 weeks of stimuli, while C57BL/6 mice show signs of heart failure within 2–4 weeks of pressure overload stimulation (100and unpublished observations). Hence strain effects need to be considered when comparing models or crossing transgenic mice with mixed backgrounds, although one can always backcross at least 6 generations to possibly obtain a more homogenous effect in the desired background.
Because genetically engineered mice have rapidly become the premier reagent for studying cardiovascular biology an investigator must keep in mind that while genetically manipulated mouse models provide a very precise means of studying genotype-phenotype relationships, the extrapolation to human disease may not be straightforward. In the context of cardiovascular biology, rudimentary physiologic function is fairly conserved between the mouse and human, but there are important differences that need to be considered. First, heart rate and systolic pressure derivatives (dP/dtmax) are considerably different between mice and humans. Average resting murine heart rates range from 550–650 beats/min verses 70 beats/min in humans99, 101 and shortening rates are 17,000 mmHg/sec in mice verses 1885 mmHg/sec in humans101. In fact, sympathetic tone overall appears to be higher in a mouse and therefore may impact cardiac reserve and in vivo function101. Second, Ca2+ handling is different in the mouse and rat relative to higher mammals such as rabbits and humans. The primary difference is that murine ventricles utilize higher sarcoplasmic reticulum Ca2+ ATPase (SERCA) activity to remove Ca2+ from the cytosol during excitation contraction-coupling, which is reflected in the compressed time scale of the rodent cardiac action potential102. In rodents SERCA contributes to 92% of the Ca2+ removal during excitation contraction-coupling with the sodium-calcium exchanger (NCX) extruding 7% and the rest outside the sarcolemma102. By contrast NCX plays a much larger role in Ca2+ extrusion in humans, contributing 28% to Ca2+ removal while SERCA sequesters the remaining 70%. Third, the primary contractile motor protein in the mouse heart is the kinetically faster α–MHC103–106 whereas humans express the slower β-MHC isoform32, 33, 104, 107. Fourth, the mouse heart also appears to have a different susceptibility to disease as eluded to in previous sections. This point is well made when one considers the genetic mouse models of HCM. Many of these murine HCM models show little to no hypertrophic growth or fibrosis, a phenotype that is very different than the clinical presentations in human populations with HCM. This is an example in cardiac research where a transgenic rabbit has been very informative in modeling HCM as it occurs in humans108. However, given the time and expense involved in generating transgenic rabbits, the mouse will probably remain as the dominant genetic system for cardiac genotype-phenotype relationships, though the rabbit can be viable alternative for transgenesis in select situations.
Transgenic and gene-targeted mouse models are frequently intercrossed to address the mechanistic function of a particular gene in the heart. While such crosses have become commonplace, it is not always clear how to prioritize amongst all the potential control groups available when crossing multiple models. Figure 2 illustrates several examples of experimental matrices of the recommended control groups for some of the models discussed in this review (Fig. 2). The number of control groups becomes more substantial with added genetic complexity, such as with a double transgenic system used for Tet-O inducible expression or Cre-loxP technology. Of course crossing multiple transgenic/gene targeted models will require single gene transgenic and non-transgenic littermates controls on the appropriate genetic background. When using the α–MHC-MerCreMer transgene other control groups need to be included, such as tamoxifen treatment to α–MHC-MerCreMer mice that are not crossed with the loxP allele-containing targeted line.
While there are non-genetic manipulations that can be used to create heart disease mimetics in larger animals that may be more generalizable to human heart disease, it is the mouse that has propelled the field of cardiovascular biology forward into a mechanistic assessment of single gene function for eventual human translational endeavors. However, properly using mouse genetic techniques, especially as the toolbox of lines and molecular manipulations continues to expand, requires all investigators to step back and reflect. We need to ensure that these tools are properly employed and that younger scientists are correctly trained in their usage, as well as to keep an ongoing dialog concerning the limitations and pitfalls underlying any potential technology or approach. Control groups also need to be fastidiously considered well in advance, prior to breeding, which can save 6 or more months in a research project. Genetic manipulation in the mouse represents a premiere approach for cardiovascular research for the foreseeable future, as no other system allows for the spatial and temporal manipulation of any gene or RNA product, with the goal of linking it as protective or causative in heart disease or other biologic processes of significance in cardiac biology.
Methodological Reviews discuss methods that are of broad interest to the community of cardiovascular investigators and that enable a better understanding of cardiovascular biology, particularly recent technologies in which the methods are still in flux and/or not widely known. It is hoped that these articles, written by recognized experts, will be useful to all investigators, but especially to early-career investigators.
Sources of funding
This work was supported by grants from the NIH (to J.D.M., J.M.M., and M.M.). J.D.M was also supported by the Howard Hughes Medical Institute. J.D. was supported by a National Research Service Award from the NIH.
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