© 2001 American Heart Association, Inc.
Editorial |
To Cre or Not To Cre
The Next Generation of Mouse Models of Human Cardiac Diseases
From the Institute of Molecular Medicine, University of California San Diego, School of Medicine, La Jolla, Calif.
Correspondence to Kenneth R. Chien MD, PhD, 0613C Basic Science Building, UCSD School of Medicine, La Jolla, CA 92093. E-mail kchien{at}ucsd.edu
Key Words: transgenic mouse models • heart disease • cardiomyopathy • gene targeting • homologous recombination
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Introduction |
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Connecting genes and molecular pathways with in vivo physiological endpoints has never been a simple task. Dissecting the effects of the causal from the phenomenal, the environmental from the inherited, the developmental from the postnatal, the primary from the secondary, and the polygenic from the monogenic has been confounding at best. For those of us interested in complex human cardiovascular diseases, the situation has been even more difficult, given the multifactorial and integrative nature of the most important human diseases and biological processes, such as angiogenesis, atherogenesis, obesity, hypertension, and heart failure, to name a few. Over the last few years, a movement to capitalize on the availability of genetically modified mouse models has led to a resurgence in integrative physiology that has been spurred on by the development of a host of new technology for assaying in vivo phenotypes in the living mouse that now encompasses conscious blood pressure recording, hemodynamic catheterization, microdigitized angiography, noninvasive imaging via echocardiography, new MRI technology, programmed stimulation, long-term Holter monitoring, microsurgical implementation of pressure overload in both the right and left ventricles, in utero myocyte implantation, and many other new techniques.1 In short, physiologists have reinvented themselves by pushing the limits of miniaturization in genetically modified mouse model systems.
In parallel, molecular biologists have expanded the tool box
for generating genetically modified animals, which now encompasses the
activation of a given mutation in specific cell types and at specific
times using cre-lox technology for conditional
mutagenesis.2 In this regard,
the study in this issue of Circulation
Research by Minamino et
al3 makes a valuable
contribution by suggesting the utility of a single transgene to allow
ligand-dependent activation of the genetic modification of interest,
which in this case is the expression of a LacZ reporter gene. The work
underscores the feasibility of jumping over embryonic lethality of a
given mutation via engineering temporal and spatial control of the
onset of the mutation of interest. Given the time, expense, and
labor-intensive nature of creating and breeding genetically modified
mouse models, this work highlights the question: to cre or not to cre?
There is a daunting spectrum of existing experimental strategies to
modify the mouse genome, each of which can be implemented with a
diverse set of promoters, targeting constructs, and ligand
activators. The pace of work in the field suggests that
many new approaches are on the immediate horizon. To provide a
framework for answering this query, a brief summary of the past,
present, and next generation of technologies for modifying the
mouse genome has been provided below, using cardiac muscle diseases as
a prototypic example (see the
Table
).
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Generation Past |
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In the beginning, there was transgenesis. Although the first transgenes resulted in the ubiquitous overexpression of a gene of interest, the cloning of a variety of cell type–specific promoters ultimately led to the identification of regulatory sequences that were capable of in vivo targeting of the expression of a given transgene to specific tissues. For example, the availability of well-characterized cardiac-specific promoters, particularly the
-myosin heavy chain
promoter (MHC),4 has led to
the generation of several informative transgenic mice, resulting in the
identification of intrinsic cardiac myocyte pathways that regulate
hypertrophy5 6
and
contractility.7
However, the high level of expression of a large subset of these
transgenes has resulted in the development of cardiac dysfunction and
cardiomyopathy that can often be attributed to a
generalized toxic effect of the transgenes, which are expressed at the
level of sarcomeric proteins. As a result, the phenotype that
reflects a primary effect of the transgene can be obscured by the
nonspecific effects of cardiomyopathy that arise
from the secondary effect of disrupting the normal stoichiometric
relationship of cardiac signaling or structural proteins. In addition,
the mosaic nature of the transgene expression, line-to-line variations
in the level of expression, effects of the genomic site of integration,
and wide differences in the copy number of the transgene can make
discrimination of primary versus secondary effects quite challenging.
Finally, gain-of-function studies do not necessarily indicate an
endogenous role for a given candidate gene in the naturally
occurring biological pathway, whereas the overexpression of dominant
negatives are confounded by their nonspecific inhibitory
effects. Thus far, the most informative cardiac transgenic genes have
been those that display a gain of physiological
function, where the observed phenotype cannot be ascribed to a
toxic effect of the transgene per se, such as the cardiac-specific
expression of an inhibitory peptide of the ß-adrenergic
receptor kinase that results in enhanced cardiac
contractility.7
In short, transgenics have been valuable as a starting point for
generating genetically modified mouse models of human disease, but the
added value of gene ablation has become increasingly
clear in identifying the role of endogenous genes in
complex cardiac physiological
endpoints.
In this regard, gene targeting has been especially valuable
in identifying genes that are involved in critical aspects of
cardiogenesis, including NKX
2.5,8
GATA-4,9 10
MEF-2,11 the HAND
genes,12 13 14
RXR,15 and several
others.16 Because the
ablation occurs via a site-specific recombination event, issues of copy
number, site of integration, and mosaicism are moot. A subset of the
most informative gene-targeted mouse models has been based on the
knockout of genes that are expressed in a cardiac muscle–restricted
fashion, including mutations in
cytoskeletal17 18 19
and calcium-cycling
genes.20 21
However, gene targeting carries with it the risks of gene redundancy
(particularly an issue for highly conserved transcription factors and
signaling molecules that have several closely related family members
that share overlapping expression patterns), inherent costs of time and
labor, and the difficulties of interpreting whether any adult
phenotype that arises actually reflects an earlier
developmental effect. Because many of the most intriguing candidate
genes are widely expressed, it can also be unclear as to whether these
genes are impacting cardiac physiology within cardiac myocytes or via
secondary effects in neighboring cell types or integrative signals from
other organ systems. Finally, and perhaps most importantly, early
embryonic lethality in conventional gene-targeted animals can prevent
an examination of the role of the gene of interest in the physiology of
the postnatal heart. These last two considerations formed the impetus
for the development of new strategies to control the onset of the gene
ablation in time and space, ie, conditional gene
targeting.
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Generation Present |
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The latest wave of mouse models has advanced beyond generalized gene knockouts to develop new strategies for the precision engineering of endogenous genes within specific cell types. The most successful approach has been based on the generation of mice that harbor floxed alleles, which contain LoxP recognition sequences that flank a critical exon that is required for the expression or function of the gene of interest2 (see the Figure
).
These mice are generated by homologous recombination of targeting
vectors in embryonic stem cells that bring in the LoxP sites into the
germ line. The floxed allele mice express the normal gene
product, because the LoxP sites are located within the intron
sequences that are spliced out during RNA processing. However, the
intervening sequences between the LoxP sites can be excised by the
expression of CRE recombinase, which is brought into the genetic
background of the floxed allele mice via interbreeding. By
controlling the expression of CRE recombinase to a specific tissue, for
example to the ventricular chamber, it is possible to
generate mice that harbor a ventricular-restricted mutation
in a gene that is widely expressed, thereby allowing a direct
examination of the role of a given gene within cardiac muscle
(Figure).
The ability of the cre mouse to drive high-level, uniform expression of
the cre recombinase and to maintain strict tissue specificity is
critical, and several promoters have been developed that will allow the
generation of cardiac-specific mutations using cre-lox
strategies.22 23 24
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The most sought after prize has been the development of approaches to control both the spatial and temporal onset of the gene modification of interest. Earlier studies have documented the ability of triggering the gene ablation with specific ligands that control the expression of a particular cre transgene that is introduced into the background of a given floxed allele. Previous studies have generated systems where the mutations are triggered by the administration of tetracycline,25 interferon,26 and ecdysone,27 to name a few. However, the challenge has been in combining both tissue specificity and inducibility into the paradigm for conditional gene modification. The first-generation strategies required the introduction of 2 transgenes (one designed to control temporal activation and the other to control tissue specificity) into the background of homozygous floxed allele mice, making the breeding strategies complex, cumbersome, time-consuming, and extremely expensive. Recently, an ingenious series of transgenes has been developed that allows both inducibility and tissue specificity in a single transgenic line. Li et al28 have generated the first of these prototypes, where CRE expression is triggered by the administration of tamoxifen to the intact animal. The expression of an estrogen receptor–CRE fusion gene is controlled with a tissue-specific promoter, whereas the CRE activity is unleashed by the administration of the ligand. The present study by Minamino et al3 provides direct evidence for the feasibility of extending this concept to postnatal myocardium, documenting the ability to maintain cardiac specificity and inducibility. Accordingly, there is now a way forward for generating conditionally mutant mice in both space and time. The growing availability of a wide variety of floxed allele mice suggests that the utility of these mice could be considerable for the field of molecular cardiology.
As with all cutting-edge work, there are several open
questions that will need to be addressed. Of particular relevance will
be the ability to drive the high efficiency of the recombination event
in endogenous floxed alleles, which will ultimately
require taking out both alleles in a sufficient number of myocytes
to exert a physiological effect in the intact organ
in vivo. To date, the efficiency of the conditional mutations has been
a recurring problem, making negative results a frequent,
uninterpretable event. Activating a reporter transgene is likely to be
considerably easier than driving the excision of authentic floxed
alleles. A direct test of this progesterone-dependent CRE
activation mouse versus other inducible cre lines is warranted to
completely validate the strategy. Because the progesterone CRE fusion
protein mouse is a transgenic animal, mosaicism could become another
critical issue, which has been found in other transgenic cre lines.
Furthermore, as noted within the data presented in the
present study, there seems to be a significant activation of the
reporter gene, even in the absence of the in vivo administration of the
progesterone ligand. This leakiness of CRE expression could complicate
the interpretation of the physiological versus
developmental role of a given gene modification and could also raise
the specter of embryonic lethality in the presence of the homozygous
floxed allele background. Whether this reflects activation from
endogenous circulating progesterone or an inherent
leakiness in the construct per se attributable to the site of
integration is worthy of additional examination. In this regard, Li et
al28 have recently
documented the feasibility of modifying the ligand activation domain
such that endogenous ligands are incapable of activating
the cre recombinase, thereby allowing a tight regulation of expression
with little or no leakiness in the basal state, a critical
consideration for gene modifications that would be expected to be
embryonic lethal. Finally, the recent observation that nonspecific
cardiac injury and cardiomyopathy can accompany the
-MHC driven overexpression of green fluorescent
protein29 and several other
genes30 raises the question
as to whether progesterone CRE fusion protein will result in cardiac
injury and dysfunction at baseline. It should be noted that previous
studies with animals overexpressing -MHC CRE have indeed documented
cardiomyopathy in a subset of lines, additionally
emphasizing this point. Nevertheless, the present study bodes well
for those interested in conditional mutations that extend from
presently established cre lines for achieving high-efficiency
cardiac-restricted conditional gene mutations.
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Generation Next |
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TopIntroductionGeneration PastGeneration PresentGeneration NextReferences |
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To cre or not to cre? Ironically, the answer may ultimately lie not in technology but in biology, ie, the nature of the question of interest. As noted above, there are a growing number of tools to modify the mouse genome, with the promise of many more on the horizon. The "generation next" of mutant mouse models is likely to include new approaches for somatic and germ-line gene rescue of conventional knockout animals, including new strategies where the knockout and rescue are encoded in a single targeting construct and triggered by a single switch. Double knockout mice have already been effectively used for true genetic complementation studies to identify new targets for heart failure progression.21 New smart vectors that will allow high-efficiency, tissue-tropic, and long-term somatic gene transfer of CRE or downstream genes that rescue the knockout phenotype are also in the pipeline. New mutant mouse models are appearing from chemical mutagenesis screens, and libraries of targeted embryonic stem cell lines and a host of floxed allele mice are becoming available from numerous laboratories. Ironically, the astounding results of the human and mouse genome projects underscore the remarkable conservation of their respective genomes, and new candidate genes are arising regularly in studies of other model organisms, including zebrafish, Drosophila, and Caenorhabditis elegans. Given the difficulty of establishing cause-and-effect relationships between given complex genotypes and in vivo physiological phenotypes in human populations, it is becoming increasingly likely that the molecular pathways will initially be identified in the mouse and subsequently corroborated in candidate gene studies in human populations. If such is the case, perhaps the well-bred mouse may eventually become man’s best friend.
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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