(Hypertension. 1996;27:495-501.)
© 1996 American Heart Association, Inc.
Articles |
Strategies for Studying Cardiovascular Phenotypes in Genetically Manipulated Mice
From the Department of Medicine, Center for Molecular Genetics and American Heart Association Bugher Foundation Center for Molecular Biology, University of California, San Diego, La Jolla.
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Abstract |
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 Top Abstract IntroductionTransgenesis and the Myocardium...Regulation of Transgene...Manipulation of the Endogenous...Reporter Genes Troubleshooting Conclusions References |
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Abstract Unraveling the pathogenesis of complex cardiovascular diseases, such as hypertension, requires the development of in vivo animal model systems. Although large-animal models have long served as the gold standard, recent advances in transgenic and gene-targeting approaches, mouse genetics, and microsurgical technology are initiating a revolution that has led to the unexpected coupling of in vivo molecular physiology with genetically engineered mice. This article discusses the design of strategies to study complex cardiovascular phenotypes in genetically modified mice, including both transgenic and gene-targeted animals. At this time, a number of strategies are used to address specific molecular or physiological questions, and examples are briefly highlighted. In addition, a number of potential problems in the generation and use of transgenic mice in the study of cardiovascular biology are presented.
Key Words: models, cardiovascular • animals, transgenic • mice, knockout • gene targeting • mice, inbred strains • myocardium
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Introduction |
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TopAbstractIntroductionTransgenesis and the Myocardium...Regulation of Transgene...Manipulation of the Endogenous...Reporter Genes Troubleshooting Conclusions References |
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Most human disease is polygenic, with several genes interacting with environmental influences leading to the clinical phenotype. Diseases generated by a single gene defect are often subject to the influence of modifiers or interacting genes that alter the expression of the phenotype and thus contribute to heterogeneity of human disease. Nowhere are multifactorial pressures more evident than in cardiovascular medicine. Hypertension and atherosclerosis clearly have a polygenic basis, and the variability of phenotypic expression in human hypertrophic cardiomyopathy is of considerable interest. Preclinical diagnosis and therapy in such disease has, until recently, not been approached by addressing the underlying molecular basis of the phenotype. Models defining cardiovascular physiology have long relied on large animals, which have inherent limitations determined by genetic variance and inaccessibility to molecular-genetic manipulation. For these and other reasons, several investigators have turned to the mouse as a model system in which to study cardiovascular disease.
The powerful methods developed for the mouse model system allow an attack on any defined biological question through several approaches (for a review, see Reference 1). Many inbred strains of mice have been produced, and within each strain, genetic variability is essentially eliminated. Phenotypic analysis in conjunction with molecular examination and/or genomic manipulation within inbred strains allows precise determination of the role of specific gene products. Studies using different genetic backgrounds in different inbred strains have allowed identification of modifiers and isolation of genes that encode interacting gene products.2 The determination of the physical map of the mouse will soon produce molecular markers at short intervals spanning the entire genome, providing an ideal vertebrate system for molecular-genetic analysis of complex traits.
Characterization of an allelic series at any particular locus is the classic genetic method used to analyze gene function. Such approaches encompass a range of mutations at a single locus, from a complete loss of function (null alleles) to various levels of reduced function (hypomorphic alleles) to new functions (neomorphic alleles; eg, constitutively active gene). In the mouse, all types of alleles can be produced by molecular manipulation of endogenous loci as well as by overexpression of wild-type or mutant transgenes in a systemic, tissue-specific, or ectopic fashion. Recent reviews have described the broad use of transgenic mice to study cardiovascular biology.3 4 The purpose of this article is to supplement these reviews and briefly describe a few examples in which the power and elegance of the mouse system have been used to address questions of development and disease of the cardiovascular system.
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Transgenesis and the Myocardium |
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TopAbstractIntroductionTransgenesis and the Myocardium...Regulation of Transgene...Manipulation of the Endogenous...Reporter Genes Troubleshooting Conclusions References |
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Analyses of the mechanisms producing particular cardiovascular phenotypes in the mouse have recently relied on overexpression of either wild-type or mutant cDNAs to produce neomorphic alleles. The optimal approach in the production of a transgenic animal is determined by the desired effect, the target tissue, and the availability of appropriate regulatory sequences to drive transgene expression. Systemic expression (ie, throughout the entire animal) is used when it produces the preferred distribution, when tissue specificity is not an option, or when this pattern of expression is not deleterious and/or will not complicate the outcome. Both broad-based (ie, expression limited to several tissues) and systemic transgene expression have been used to elevate the serum levels of the wild-type isoform of several components of lipoprotein transport metabolism (Table 1
).
Overexpression of human apolipoprotein
AI5 6 or rat apolipoprotein E7 in
transgenic
mice appears to provide protection against diet-induced formation
or progression of atherosclerotic lesions. Species-specific
differences in human and mouse lipoprotein metabolism argue
against acceptance of absolute validity for such models,
yet these transgenics provide direct evidence that increased HDL levels
protect against atherosclerosis.
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Systemic overexpression of transgenes may complicate the interpretation of phenotypes within individual transgenic animals. It is therefore necessary in many cases to limit transgene expression to individual tissues or cell types. This approach was used to produce high levels of cardiac-specific expression of the ß2-adrenergic receptor,8 ß-ARK, or a ß-ARK inhibitor.9 10 The resulting increased density of ß2-receptors produces an increase in myocardial contractility at rest, suggesting that activated ß2-receptors can couple to G proteins in the absence of agonist. Furthermore, Bond et al9 showed the first in vivo evidence for the existence of inverse agonists (ie, molecules that turn off precoupled receptors) with the potential to elucidate clinically important therapeutic agents. Tissue-specific overproduction of ß-ARK and ß-ARK inhibitor also suggests that these molecules are important in regulating myocardial contractility. Increased concentrations of ß-ARK did not affect basal contractility, but they caused a reduction in the isoproterenol-induced response, whereas overexpression of a ß-ARK inhibitor increased basal as well as isoproterenol-induced myocardial contractility.
Expression of wild-type proteins in tissues in which they are not normally found provides the means to assess the role that a factor may have in the determination of the fate of a particular cell. It is well known that the four members of the MyoD family of HLH transcription factors are essential for skeletal muscle development. However, the heart is apparently normal in mice that lack individual or pairs of the HLH proteins.11 12 Although these genes are not expressed in wild-type murine heart (for a review, see Reference 13), Litvin and coworkers14 reported the presence of an HLH protein in the early heart primordia of the chick. In an attempt to show that MyoD and Myf-5 can initiate skeletal myogenesis in vivo, each factor has been ectopically expressed in the heart of transgenic mice. Production of MyoD in the heart stimulates expression of some skeletal muscle–specific genes.15 Partial initiation of skeletal myogenesis in the heart causes abnormal cardiac development and fetal death. Expression of Myf-5 in the heart stimulates the genes observed early in skeletal myogenesis and leads to myofiber degeneration with cardiac dilatation,16 indicating that these transcription factors can initiate some of the skeletal muscle program in the cardiac context that is not compatible with proper heart development.
Targeted expression of mutant proteins in the cardiovascular system of the mouse has the potential to elucidate the determinants of individual physiological phenotypes in both the adult and the developing animal. The production of oncogenic ras in the ventricles of the mouse heart, for example, stimulates hypertrophy in the adult with an impairment of diastolic function17 similar to the clinical phenotype of human hypertrophic cardiomyopathy. During early embryonic development, overexpression of a dominant negative form of the tyrosine receptor kinase tek results in mid-gestational death, which could be due to either severe disruption of endothelial tissue, vascular leak and hemorrhage, or abnormal heart development.18 Embryos deficient for tek produce endothelial cells, but expansion of this cell population fails to occur, suggesting the tek is required for the maintenance of the endothelial cell fate. It remains to be shown whether the myocardial defect derives from a missing interaction with the developing endothelium or is secondary to a defect in the endothelial cells.
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Regulation of Transgene Expression |
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TopAbstractIntroductionTransgenesis and the Myocardium...Regulation of Transgene...Manipulation of the Endogenous...Reporter Genes Troubleshooting Conclusions References |
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Potentially the most powerful as well as the most limiting aspect of generating transgenic animals is the promoter sequences used to regulate expression of the transgene. Production of transgene product in the preferred spatial pattern, at an appropriate time, and at effective concentrations is characteristic of ideal regulatory sequences.17 The transgenic promoter should limit expression to the tissue, region, or cell type of interest. Very few tissue- or cell type–specific promoters target expression to the cardiovascular system (for a review, see Reference 3). In some cases, it may be possible to use a subfragment of the full-length promoter to drive tissue- and/or time-specific expression. For example, the endogenous MLC2v protein is present in both slow skeletal muscle and ventricular myocardium19 ; however, a small fragment of this promoter limits expression to the ventricular compartment in transgenic mice.20
The ideal promoter should provide appropriate developmental regulation.
The temporal regulation of a transgenic promoter can potentially limit
usefulness in addressing particular questions. The 5.5-kb
-myosin heavy-chain promoter has been used extensively to
produce expression throughout the entire myocardium in
adult animals (eg, ß2-receptor mice), yet during
development this gene is shut off in the ventricles,21
indicating that it would have limited use in experiments addressing
mid- to late-gestational ventricular development, the
time at which most cardiogenesis occurs. In fact, for some
cardiovascular questions the available
cardiac-specific promoters may not produce the desired temporal
pattern of expression. Most of the promoters currently used to limit
transgene expression to the heart are temporally expressed early in
development and continue throughout the life of the mouse. Transgene
expression throughout the life of the mouse could complicate the
observed phenotype—for example, leading to early developmental
defects or to compensatory effects in the adult—and might not produce
the desired model intended for dissection of primary molecular
mechanisms of cardiovascular disease. Such limitations
have spawned the production of controllable promoters that
yield inducible transgene expression.
The ability to control gene expression facilitates analysis of tissue-specific gene function and provides the means to generate timed developmental defects or to determine the effects of transgene expression limited to the adult. Promoters inducible via metal ions, heat shock, or steroid hormones can drive low levels of transcription even in the absence of inducers and therefore suffer from the lack of stringent regulation. A system inducible by tetracycline or its analogues has extremely low levels of basal transgene expression, which can be stimulated up to 5000-fold and still retain tissue specificity.22 This system holds great promise for tissue-specific inducible transgene expression, yet cardiac-specific inducible expression in vivo has yet to be achieved.
Finally, promoter strength should be sufficient to produce active
amounts of the transgene product. Although 250 bp of the MLC2v
promoter produces ventricle-specific expression, RNA levels
produced from this fragment are low (J. Hunter and K. Gottshall,
unpublished data, 1995). This may be an advantage when high levels of
transgene expression may lead to a lethal phenotype. In most
cases, however, the desired level of transgene expression is at or near
levels of the endogenous counterpart of the transgenic
promoter being used. Both the human cardiac actin23 and
mouse -myosin heavy-chain promoters24 can
produce high levels of transgene RNA in the heart. The human cardiac
actin promoter can produce amounts of RNA equivalent to the
endogenous mouse gene; however, this high expression
requires an enhancer element that is located 3' to the human actin
coding sequences. To further increase expression levels 10- to
100-fold, an intron can be added to the transgene construct between the
promoter and the cDNA.25 26 For any particular gene,
it
may be necessary to study several transgenic animals, each taking
advantage of the expression patterns of different promoter sequences to
address both developmental and adult-related questions.
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Manipulation of the Endogenous Locus |
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TopAbstractIntroductionTransgenesis and the Myocardium...Regulation of Transgene...Manipulation of the Endogenous...Reporter Genes Troubleshooting Conclusions References |
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Homologous recombination can be used to manipulate an endogenous locus in ways that may overcome several of the concerns one has when using transgenic promoters. Homologous recombination is used to replace sequences at the genomic locus with DNA that has been altered in vitro (for a review, see Reference 27). This process is accomplished in totipotent embryonic stem cells28 derived from the inner cell mass of a mouse blastocyst. Under the appropriate culture conditions, embryonic stem cells remain undifferentiated and subsequently can contribute to all cells of an adult when injected into the blastocele of a developing embryo. The resulting animal has an inbred genetic background harboring a single change at the targeted locus. This method makes it possible to eliminate or modify endogenous gene function creating duplications, deficiencies, or hypomorphic or neomorphic alleles.
Hypertension is a polygenic disease in which both clinical and physiological studies and genetic mapping have implicated the involvement of the renin-angiotensin system.29 30 The genes encoding angiotensinogen and ACE have been analyzed by use of gene targeting to produce both deficiencies and duplications at the endogenous mouse loci. This approach does not alter the regulatory elements of the locus, and analysis can be accomplished in a homogenous genetic background.31 The angiotensinogen locus was altered to produce a duplication as well as a deficiency in separate strains of mice.32 Interbreeding allowed production of an allelic series of animals with zero, one, two, three, or four copies of the wild-type gene. Kim and colleagues32 show that blood pressure changes directly in response to gene dose, establishing a causal relation between angiotensinogen and blood pressure regulation. Even though genetic studies provide conflicting reports as to the linkage of the gene encoding ACE,29 33 Krege et al34 supply clear evidence that ACE has a role in the regulation of blood pressure. Male mice hemizygous (+/-) or deficient (-/-) for the ACE gene show decreases in blood pressure commensurate with gene dose and have decreased fertility. Female mice display normal fertility, and only the animals deficient for ACE have decreased blood pressure. Both of these studies show significant changes in blood pressure that are relatively easy to detect. Mating these animals to mice of various genetic backgrounds could lead to the discovery and analysis of interacting genes involved in blood pressure regulation, making these useful models for the study of essential hypertension.
For many genes, the knockout or complete removal of function throughout the entire animal may produce an early lethal phenotype, thus complicating subsequent analysis. Deletion of an individual gene restricted to a tissue and/or time in the life of a mouse can be created by use of the site-specific Cre recombinase of bacteriophage P1.35 This system requires the generation of two lines of mice. First, the gene of interest is manipulated in vitro to include Cre recognition sequences (called loxP sites) flanking functionally required exons. Subsequent homologous recombination inserts this construct at the endogenous locus. The other mouse line is a standard transgenic animal containing the Cre recombinase cDNA under the control of a tissue-specific promoter. Expression of Cre recombinase in a mouse homozygous for the loxP targeted locus removes the genomic DNA internal to the loxP sites, eliminating gene function. Regulating the transgenic expression of Cre recombinase with a spatially and/or temporally controlled promoter generates conditional, cell type–specific gene knockout. Although this approach is yet to be accomplished in the cardiovascular context, it has been used to create a T-cell–specific deletion of the ubiquitous enzyme DNA pol-ß, involved in DNA repair.35 loxP sites were added to the pol-ß locus via recombinant DNA and homologous recombination methods. Cre recombinase expression was regulated by a T-cell–specific promoter in transgenic mice. In animals heterozygous for the targeted pol-ß gene and hemizygous for the Cre transgene, deletion of the pol-ß fragment was observed in 63% to 84% of the splenic T cells. These experiments appear to be limited by the transient expression of the promoter producing Cre, and in other situations it will be prudent to maximize recombinase expression to achieve 100% deletion frequencies. This study brings forth the very exciting possibility of circumventing a lethal phenotype and being able to systematically examine lack of gene function in any tissue or tissues of interest. One limitation of this system occurs because most genes are expressed at some time during embryonic development, and tissue-specific knockout will occur as soon as the recombinase is produced. This may be overcome through cis-acting control elements that confer inducible regulation to the promoter driving Cre expression, such as the tetracycline-controlled system mentioned above.
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Reporter Genes |
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TopAbstractIntroductionTransgenesis and the Myocardium...Regulation of Transgene...Manipulation of the Endogenous...Reporter Genes Troubleshooting Conclusions References |
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Nearly all of the experiments discussed so far rely on prior knowledge of the tissue and temporal patterns produced by the promoter used in each transgenic animal. In situ hybridization using antisense RNA probes in whole mount or tissue sections is the standard for the determination of such patterns. Although extremely informative, this technique is laborious, complex, and highly sensitive to background distortion. It has now become commonplace to use reporter genes in transgenic mice to explore the issues of promoter-generated pattern as well as to discover the cis-acting elements responsible for transcriptional activity. The key elements in the usefulness of any reporter gene are the sensitivity and ease of detection (Table 2
). Nearly any gene can be used as a reporter (for a
review, see Reference 36), but detection may be limited to in situ
hybridization with specific probes. The growth hormone genes are an
example of a marker that can be detected only by in situ hybridization
with nucleic acid probes37 or by immunohistochemistry with
species-specific antibodies.38 The use of this
reporter is further complicated by the biological activity that growth
hormone may have on the target tissue. Single-cell detection of
firefly luciferase RNA is also limited to hybridization or
immunohistochemical methods; however, the ability to detect this marker
in cell extracts makes it an extremely useful measure of gene
expression.20 39 Similarly, the gene encoding CAT is
used
to analyze gene expression in cell extracts from transgenic
animals or from cells in culture.40 The luciferase assay,
a quantitative measurement of photon emission, is considered more
sensitive than the CAT assay, which depends on enzymatic
acetylation of substrate and subsequent thin-layer
chromatography followed by
autoradiography. Recent methods have been developed
to increase the sensitivity of the CAT assay to rival that of
luciferase.41 Furthermore, a histochemical procedure
recently described allows in situ detection of CAT protein by the
formation of a brown precipitate in cells expressing the
transgene.42
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The most popular marker in the study of temporal and spatial gene expression is LacZ. This enzyme catalyzes the hydrolysis of 5-bromo-4-chloro-indolyl-ß-D-galactoside (X-gal) to form a blue precipitate that is visible at the single-cell level43 44 and can be detected in a quantitative assay from cell extracts.45 The detection of this marker consists of tissue-fixation and the addition of chromogenic substrate, a simple process that generally produces high-resolution results. However, LacZ is not without its share of problems. Mammals have an endogenous ß-galactosidase activity that can contribute to background staining, although this is not present in the heart. There are pH differences for optimum activity between the mammalian and bacterial enzymes that can be used to reduce the level of background, yet even at high pH, the mammalian protein may display residual function. Differentiation of the endogenous and transgene staining can be achieved by cloning a nuclear localization signal into ß-galactosidase.46 47 48 Subsequent tissue staining will produce blue nuclei in cells expressing the transgene, whereas the endogenous signal will be cytosolic. It has been observed that sequences in the LacZ cDNA can produce unexpected distribution of transgene expression. A cell type–specific neural promoter driving expression of LacZ produces ß-galactosidase in neurons that do not express the endogenous gene.49 However, the same promoter expresses a different marker in a pattern that matches the endogenous gene. Finally, results of LacZ expression postnatally and in adult tissues do not always produce predictable expression patterns (for a review, see Reference 36), the reasons for which are not understood.
Recently a new marker has come to light that allows identification and analysis of living cells expressing this protein. The cDNA encoding the GFP from the jellyfish Aequorea victoria has been cloned.50 This small protein (26 900 kD) functions as a monomer and emits green light in response to long-wave UV stimulation in either living or formaldehyde-fixed tissue. GFP functions as a fusion protein and has had no deleterious effects on the function of the protein it is fused to.51 This marker can be detected in Dictyostelium (for a review, see Reference 52), plants (for a review, see Reference 53), Caenorhabditis elegans,54 Drosophila (for a review, see Reference 55), mammalian cells (for a review, see Reference 56), and transgenic mice.57 Mutations at the amino acids that constitute the chromophore transform GFP into a brighter molecule with absorption and emission spectra suited for standard filter sets used in the detection of fluorescein isothiocyanate. Cells transfected with GFP expression plasmids have been purified by fluorescence-activated cell sorting.58 This should make it possible to purify any cell population from transgenic mice, or any other organism, in which GFP is expressed in a tissue-specific fashion. As was suggested for the choice of transgene promoter, the choice of marker will depend on the application, and any one reporter gene most likely will not suffice.
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Troubleshooting |
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TopAbstractIntroductionTransgenesis and the Myocardium...Regulation of Transgene...Manipulation of the Endogenous...Reporter Genes Troubleshooting Conclusions References |
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The use of transgenic mice to study cardiovascular biology has relied on advances in rDNA as well as the ability to make physiological measurements in small animals. Microsurgical approaches have been developed to produce models of either right or left ventricular dysfunction via pulmonary artery or aortic constriction, respectively.59 60 In these models, quantitative analysis of ventricular function has been measured by x-ray contrast microangiography. These techniques as well as methods for determination of respiratory function are impressive but are invasive, requiring anesthesia to produce high-resolution results, and may result in the death of the individual (for a review, see Reference 61). Noninvasive techniques such as echocardiography can be used to measure chamber size, wall thickness, and cardiac function (Reference 62; Tanaka N., M.D. et al, 1995, unpublished data); however, the level of resolution presently attainable must be improved if this method is to be of widespread use. Efforts to miniaturize technology for assessment of cardiac physiology must progress to achieve full advantage of the molecular genetics available in the mouse system in the study of cardiovascular biology.
The temporal and spatial patterns generated when promoter fragment is
used to drive expression of a transgene are of prime importance in
transgenic technology. Assuming that the transgene will be expressed in
exactly the same distribution as the endogenous gene may
lead to problems in the analysis of transgenics. Experiments
involving the atrial natriuretic peptide promoter show
differences between in vitro and in vivo regulation in response to
-adrenergic stimulation.63 Similarly, discordance
of transgene and endogenous gene expression patterns has
been described, for example, in the tek regulatory
sequences.64 The most likely explanation is that the
promoter fragment does not include all of the cis-acting
sequences necessary to mimic the endogenous distribution.
Alternatively, reporter gene analysis may bring to light
previously unrecognized patterns of endogenous gene
expression. The MLC 3f gene was thought to be expressed only
in skeletal muscle; however, Kelly et al65 discovered that
this promoter will produce ß-galactosidase in the heart.
Subsequent in situ hybridization with a probe to the
endogenous gene showed that this gene is active in the
heart.
Transgene regulation may be affected by the copy number and/or the site of chromosomal integration. Transgene insertions can be complex events consisting of one or many copies at any particular location and may contain chromosomal translocations as well. It is common that only a percentage of the transgenic animals generated (ie, founder lines) will express the exogenous gene, even though all of these animals may contain the intact gene. Bonnerot et al46 have shown position-dependent effects on temporal and spatial distributions of transgene expression in analysis of several different transgenic lines harboring the same promoter/reporter construct.
Embryonic or neonatal death is the phenotype commonly observed in many knockout lines and transgenic lines (eg, tek null and ectopic expression of myogenic factors in the heart). If death is not the result, then often it is difficult to discriminate between the developmental and physiological effects. Interpretation, for example, of the atrial natriuretic peptide knockouts as well as the overexpression of ß2-receptors may be confounded by systemic and/or tissue-specific compensatory changes that occur in response to chronic lack of function or overexpression, respectively. This problem should be eliminated with the future use of controllable transgenes as well as inducible time- and/or space-specific gene knockouts.
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Conclusions |
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TopAbstractIntroductionTransgenesis and the Myocardium...Regulation of Transgene...Manipulation of the Endogenous...Reporter Genes Troubleshooting Conclusions References |
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Current technology in molecular genetics of the mouse provides a powerful system to explore the physiological role of wild-type and mutant molecules. Mouse models of human disease allow access to underlying molecular mechanisms. Physiological differences between mice and humans do present some minor limitations. However, even if the mouse does not have the molecular counterpart of a human protein, valuable information regarding function in normal and/or disease processes can be obtained in transgenic mice. Proper use of homogeneous genetic background minimizes phenotypic variability and enhances the prospect of mapping genes encoding proteins that interact in the generation of disease. This opens the mammalian genome to a level of analysis previously unprecedented in history.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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Dr Becker is supported by an individual National Research Scholar Award from the National Institutes of Health (NIH), Dr Gottshall is supported by a training grant from the NIH and National Eye Institute, and Dr Chien is supported by grants from the NIH/National Heart, Lung, and Blood Institute and American Heart Association. The authors would like to thank Dr Andrew Grace, Dr Peter Gruber, and Dr Howard Rockman for their critical comments and discussions during the preparation of this paper.
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Footnotes |
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Reprint requests to Kenneth R. Chien, MD, PhD, Professor of Medicine, Department of Medicine, 0613-C, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA 92093. E-mail kchien@ucsd.edu.
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TopAbstractIntroductionTransgenesis and the Myocardium...Regulation of Transgene...Manipulation of the Endogenous...Reporter Genes Troubleshooting Conclusions References |
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