© 1998 American Heart Association, Inc.
Mini Review |
Timing Is Everything in Life
Conditional Transgene Expression in the Cardiovascular System
From the Albert Einstein College of Medicine, Bronx, NY.
Correspondence to Glenn I. Fishman, MD, Cardiovascular Institute, Department of Medicine, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1269, New York, NY 10029-6574. E-mail glenn_fishman{at}smtplink.mssm.edu
Abstract
Abstract—Manipulation of the mouse genome by traditional transgenic approaches has facilitated studies of gene function within the context of the intact organism and allowed for the creation of useful animal models of human disease. However, the timing of gene activation or repression is a critical determinant of phenotype, and the ability to regulate the temporal profile of transgene expression remains an important experimental goal. In this Mini Review, we describe the current status of systems to tightly regulate target gene expression in vivo, focusing on binary systems using chimeric transcription factors. Although experimental difficulties persist, regulated expression systems are beginning to produce conditional phenotypes with exciting experimental implications. We review the experience to date and examine the potential utility of these approaches within the context of cardiovascular medicine.
Key Words: mouse genome • gene expression • chimeric transcription • transgenic • conditional
Overview
Phenotype reflects the precise hierarchical control of gene expression. From a purely descriptive point of view, gene expression in multicellular organisms may be defined in terms of its spatial and temporal characteristics. Modulation of either parameter may lead to phenotypic sequelae of varying importance. Although considerable progress has been made identifying and characterizing the cis- and trans-acting factors that regulate transcription, our present understanding of gene expression is in general inadequate to functionally separate those elements that individually control the timing and location of gene expression.1 Pragmatically, then, the experimentalist typically selects regulatory elements with appropriate tissue specificity and learns to live, often unsatisfactorily, with the magnitude and timing of expression within the tissue of interest.
A detailed review of the many approaches available for conditional transgene expression has recently appeared.2 Therefore, in this Mini Review, we first focus on a consideration of experimental scenarios that provide the impetus for the continued development of conditional transgenic systems. We then describe the experience to date with several regulated systems, highlighting the tetracycline-regulated binary system that has emerged as the present system of choice for a number of investigators.3
Experimental Paradigms
We can envision models of at least four types in which regulated transgene expression may be desirable, ie, models in which (1) toxicity is observed with constitutive expression using either viral or cellular promoters, (2) potential stage-specific effects of transgene expression are investigated, (3) the relationship between transgene abundance and phenotype is examined, and (4) transgene expression is reversibly controlled. We discuss each of these paradigms in greater detail below.
Toxic Gene Products
Increasing numbers of investigators are generating transgenic mice
with dysregulated expression of wild-type or mutationally engineered
gene products in the heart, often using the well-characterized
mouse 
-MHC promoter.4 As many as 30 new models
were presented at a recent meeting of
cardiovascular scientists.5
Although occasionally the goal is to examine the function of a
previously uncharacterized gene product, more often the
investigator wishes to understand the consequences of dysregulated
expression of a protein with known activity. In many instances, the
goal is to generate a mouse model of a clinically relevant human
disease syndrome, where alterations in expression of the gene in
question are hypothesized to contribute to the pathophysiology. Not
infrequently, such efforts result in early lethal phenotypes,
such as the intense inflammatory myocarditis observed with
overexpression of tumor necrosis factor-, the massive atrial
dilatation and perinatal mortality associated with overexpression of
the type 1a angiotensin II receptor, or the disruption of
normal looping morphogenesis and embryonic lethality observed with
constitutively activated transforming growth factor-ß
receptor mutants.6 7 8 From a purely pragmatic
point of view, early lethality precludes the establishment of
transgenic lineages, necessitating the de novo generation of transgenic
founders for any additional experimentation. More important, these
outcomes prevent examination of the consequences of dysregulated gene
expression in the context of the adult organism, where most forms of
acquired cardiovascular disease are manifest.
Stage-Specific Transgene Expression
In most mammalian tissues, profound developmental alterations in
gene expression are observed; this phenomenon has been especially well
explored in the heart, where the transition from the so-called
"fetal" to "adult" program of transcriptional activity has been
extensively characterized.9 10 11 12 Inasmuch as the
cellular environment is undergoing profound ontogenic modulation, the
effects of ectopically expressed genes are likely to vary as a function
of developmental stage. For example, dysregulated expression of a
mutationally activated retinoic acid receptor in the heart
under the control of regulatory elements from the -MHC isoform is
without obvious phenotype, whereas earlier activation in the
ventricular myocardium using ß-MHC regulatory
sequences leads to profound effects on cardiac structure and
function.13 Recently, when a conditional strategy
was used (described more fully below), it was shown that induction of a
mutationally activated protein kinase C ß-isoform in the
neonatal heart leads to sudden death, whereas activation beginning in
adult mice is better tolerated, with modest concentric
ventricular
hypertrophy.14 Finally,
phenotypes observed in late stages, such as the abnormal
multinucleation and sustained DNA synthesis observed in adult cardiac
myocytes overexpressing cyclin D1, may be predicated on the perinatal
expression observed with the -MHC promoter.15
Together, these studies highlight the concept that susceptibility to
the phenotypic effects of various gene products may be stage
specific and underscore the need for tools to more fully define the
precise windows of vulnerability.
Manipulation of Transgene Abundance
Gene dosage may profoundly influence phenotype. Whereas
transgene expression may not necessarily correlate with copy number,
phenotypic severity frequently does reflect abundance of the transgene
product. For example, overexpression of transcription factors may
result in transcriptional squelching and unanticipated effects on gene
expression.16 Similarly, the abundance of
mutationally activated or dominantly negative polypeptides may
potently influence phenotype. Consider the incorporation of a
mutant dominant-negative polypeptide into a multimeric
structure,17 such as a potassium channel formed
by the association of four -subunits.18 If all
channels with at least one mutant subunit are nonfunctional, the
current (I) may be described by the following equation:
I=(1-fmut)4 xIwt,
where fmut is the fraction of mutant subunits,
and Iwt is the wild-type
current.18 The relative abundance of the mutant
protein reduces function (ie, current) dramatically over an extremely
narrow window. Thus, quantitative control of the magnitude of mutant
protein expression renders the extent of ionic flux amenable to precise
regulation and greatly enhances the power of the experimental paradigm.
These observations suggest that animal models in which phenotypic
severity is predicted to vary in a quantitative manner with respect to
the abundance of the transgene might be ideally suited for study using
regulated gene expression systems.
Reversibility
A major impetus for transgenic and gene-targeting strategies is
the development of suitable mouse models of human disease states.
Whereas gene disruption and gene replacement strategies are appropriate
to create null or gain-of-function mutants, neither is ideal to model
"acquired" diseases, ie, those with polygenic etiologies and
environmental influences. Gene-targeting strategies (with the exception
of those using recombinase-based techniques, considered below) create
germline mutations, and the potential to initiate a cascade of
secondary responses during the earliest stages of development is
substantial. These responses, even if phenotypically silent in the
unstressed organism, may considerably alter behavior to additional
challenges in the adult mouse. Although this information, in itself,
may be of interest, rendering a transgene silent during development and
inducing its expression in the adult provide a potentially more
suitable environment in which its effects may be examined, an
environment not complicated by potential developmental perturbations.
Moreover, in contrast to the permanent modification of gene expression
achieved with traditional gene-targeting strategies, conditional
transgenic approaches allow one to reversibly manipulate the expression
of a transgene. Thus, one may examine not only the process of disease
progression but also the adaptive and potentially reparative mechanisms
that are used during disease regression. For example, conditional
overexpression of a Gi-coupled heptahelical
receptor has recently been reported to result in a syndrome resembling
human congestive heart failure. Repression of transgene expression,
even in the later stages of the syndrome, leads to a dramatic reversal
of the illness.19 Conceptually, it is easy to
imagine that such reversibility will facilitate dissection of the
manifold interactions that give rise to complex syndromes such as
congestive heart failure.
Conditional Gene Expression In Vivo
An ever-expanding repertoire of systems to conditionally regulate gene expression has been described; these systems are highly effective in vitro. Several of these regulatory schemes have been successfully adapted for in vivo use, and details of these paradigms have recently been reviewed.2 Inducible promoters, such as the glucocorticoid-responsive mouse mammary tumor virus long-terminal repeat, the interferon-inducible Mx1 promoter, and the heavy-metal inducible metallothionein promoter are experimentally straightforward, and relatively robust inductions of heterologous sequences may be achieved.20 21 22 Nonetheless, pragmatic difficulties and inherent theoretical limitations render use of these systems unsuitable for the majority of investigators, accounting for their failure to be more widely used. Nonrepressible baseline transcription, ie, leakiness, is a persistent technical limitation with most of these systems. For others, toxicity or pleiotropic effects of the inducing agents are problematic. More important, for almost all of the earliest systems, there is no inherent mechanism for restricted cell-type–specific expression. Although the abundance of transgene expression may be regulatable, the spatial profile is not amenable to experimental control, and tissue specificity simply reflects the intrinsic activity of the inducible promoter used. Thus, in contrast to traditional gene-targeting experiments using tissue-specific promoters, here one gains temporal control but relinquishes the arguably more important spatial regulation.
Ligand-Regulated Binary Systems
In theory, the emergence of ligand-regulated binary transgenic
systems has addressed the dual problems of spatial specificity and
temporal regulation.23 24 25 26 These systems require
two genetic elements typically integrated independently into separate
transgenic mouse strains, as schematized in Figure 1
. The first element, the
transactivator, uses tissue-specific regulatory elements,
such as the -MHC promoter, to direct expression of a chimeric
transcriptional activator in a tissue-specific manner. The
transactivator has three essential components. First, there
exists a sequence-specific DNA-binding domain with no known
endogenous mammalian target. The second component is a
ligand-binding domain, occupancy of which efficiently regulates the
ability (either positively or negatively) of the
transactivator to interact productively with its
cognate DNA binding site. Third, the chimeric protein includes a strong
transactivation domain. Expression of the transactivator in
a tissue-specific manner imparts spatial control on the system, whereas
ligand administration regulates the efficiency and hence the magnitude
of transcription. The second element in this binary system is the
target gene, consisting of a transcriptionally quiescent minimal
promoter flanked by multimerized copies of the site-specific
DNA binding site. By crossbreeding, compound heterozygous progeny that
harbor both elements of the regulatory scheme are generated, allowing
ligand-dependent and tissue-specific target gene transactivation.
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To date, most experimental work has been carried out with variants of two chimeric transactivators, based on either the lac or tet repressors of Escherichia coli.3 23 27 Taking advantage of the modular nature of most transcription factors, the DNA- and ligand-binding domains from these two proteins have been retained, whereas their transcriptional repression domains have been replaced with strong trans-activation regions from heterologous proteins, such as HSV virion protein 16. The resulting chimeric proteins, such as the recently described tTA, are thus converted from normal repressors to ligand-regulated transactivators. The tetracycline system has some theoretical and pragmatic advantages compared with lac repressor–based approaches. First, the strength of specific interactions between the tet repressor, tetO sequences, and tetracycline analogues are all greater than the corresponding interactions between the lac repressor, lacO, and IPTG. Second, unlike the exceedingly low concentrations of tetracycline required for complete transcriptional repression, substantially higher dosages of IPTG are necessary, approaching the toxic range when used in vivo.28 29
On the basis of these considerations, several laboratories, including
our own, have examined the ability of tTA to conditionally
transactivate appropriately designed target genes in the
cardiovascular system and elsewhere. Using regulatory
elements from the -MHC gene to impart cardiac myocyte-restricted
expression, we initially examined the ability of mice harboring an
-MHC–tTA transgene to successfully transactivate
coinherited luciferase or nuclear-localized ß-galactosidase
transgenes.26 These studies demonstrated the
efficacy of the binary system but also underscored the potential for
tTA-independent transcriptional leakiness, as well as the existence of
heterogeneity of expression on a cell-by-cell basis
(Figure 2). Both of these undesirable
features may primarily reflect the integration site of the target gene
rather than the behavior of the transactivator line, per
se, since independent lines harboring identically constructed target
transgenes show substantially different degrees of background
expression and also vary in the extent of spatial
heterogeneity. Importantly, for the investigator, for
most target genes we have examined, transgenic lines can be identified
with virtually no background expression and complete repression by
exogenous tetracycline analogues. To date, when this same strain of
cardiac-specific transactivator has been used, successful
transactivation of a number of different target strains has been
achieved, resulting in stage-dependent and/or reversible
phenotypes and including proteins as diverse as a mutationally
activated form of protein kinase Cß, an engineered variant of
a Gi-coupled heptahelical transmembrane receptor
and the diphtheria toxin A gene.14 19 30 This
last example is of particular interest with regard to the issue of
toxicity and tTA-independent transcriptional leakiness. Transgenic
founders harboring the tetracycline-responsive diphtheria toxin A
target gene were recovered with rather low efficiency, presumably
reflecting toxic effects from transcriptional leakiness in most embryos
injected with the construct and embryonic lethality. Nonetheless, lines
could be identified that appeared healthy and had normal viability,
including one that was appropriately transactivated by tTA in a
tetracycline-dependent manner. Thus, founders that harbor constructs
with even the most toxic of gene products may be successfully
generated with this approach.
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Initial studies examining the feasibility of conditional transgene
expression elsewhere in the cardiovascular system have
recently been reported. With a strategy similar to that described
above, tTA has been expressed using transcriptional regulatory elements
from the SM22 promoter.31 In embryos,
tetracycline-repressible transactivation was observed in the heart,
developing vascular tree, and somitic mesoderm, similar to the known
pattern of endogenous SM22 expression at this stage of
development32 (Figure 3A). In adult mice, reporter gene
expression is restricted to smooth muscle cells of the
arterial tree, as illustrated in Figure 3B. It is likely
that the ability to conditionally express target genes within distinct
lineages of the cardiovascular system will be expanded,
limited only by the specificity of regulatory elements used to drive
transcription of tTA and other chimeric transcription factors.
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Outside the cardiovascular system, several laboratories have also reported success with tetracycline-regulated transgenic models. In two instances, genetic crosses with mice harboring a tetracycline-responsive simian virus 40 T-antigen target gene have resulted in conditional cellular transformation. When an insulin promoter was used to restrict expression of tTA to pancreatic islet beta cells, binary offspring developed infrequent islet cell tumors, from which cell lines were established with tetracycline-repressible proliferation.33 A similar phenomenon was observed in submandibular glands of binary mice harboring the T-antigen target transgene and tTA under transcriptional control of the mouse mammary tumor virus long-terminal repeat.34 In a particularly interesting result, conditional expression of a mutationally activated form of calcium-calmodulin–dependent kinase II in the forebrain resulted in reversible defects in memory formation.35
Modified Tetracycline-Controlled Transactivators
Several variants of tTA have recently been generated in an effort
to expand the utility of the regulatory system, particularly with
respect to in vivo use. In a screen of random mutants within the
repressor domain, one isolate was identified that rendered the protein
paradoxically a transcriptional activator in the presence
of tetracycline analogues, in contrast to the repression observed with
tTA itself.36 Since target gene induction with
tTA requires metabolism and clearance of tetracycline,
especially from bone,37 de novo transcription
follows with some temporal delay. Thus, the so-called rtTA has some
theoretical advantages for studies when very rapid induction of target
genes is desirable.38 Although the rtTA approach
has worked well in tissue culture applications, the experience to date
in transgenics is limited to the original report describing successful
transactivation of a reporter gene using a CMV-rtTA construct. Finally,
tTA truncation mutants with graded transactivation potential have been
described.39 These variants, which have truncated
VP16 transactivation domains, may minimize interactions with
endogenous cellular transcription factors and reduce the
likelihood of potentially toxic transcriptional squelching. The utility
of these modifications for in vivo use has yet to be examined.
Hormone-Regulated Transgene Expression
Similar ligand-dependent strategies using hormone-responsive
transcriptional activators to regulate target transgenes
have been described. For example, a chimeric transactivator
consisting of a mutated progesterone receptor ligand–binding domain,
the yeast GAL4 DNA–binding domain, and the HSV VP16 transactivation
domain has been shown to transactivate appropriate target genes
only in the presence of mifepristone (RU486), a progesterone
antagonist, at concentrations substantially lower than
those that block the normal biological effects of
progesterone.40 By use of this approach,
expression of human growth hormone was conditionally expressed in mouse
liver, resulting in ligand-inducible growth
enhancement.41 The Drosophila ecdysone
receptor, which normally heterodimerizes with ultraspiracle
(a homologue of the mammalian retinoid X receptor) to mediate the
biological activity of the steroid hormone 20-OH ecdysone, has been
similarly modified, also resulting in hormone-dependent transactivation
of appropriately engineered response genes.42
Although the single study in transgenic mice suggests that
ecdysone-regulated transgene expression compares quite favorably with
tetracycline-controlled systems, to date, no reports of regulated
expression of functional genes have appeared,43
and additional experience will be required to directly compare these
two approaches. Moreover, the potential exists for inappropriate
sequestration of endogenous retinoid X receptor by the
targeted ecdysone receptor and unanticipated phenotypic
consequences.
Dimeric Ligands
To some extent, transcription factors have structurally distinct
DNA-binding and activation domains, and site-specific transactivation
may be reconstituted by close physical approximation of these two
independent domains. This association may be facilitated by fusion of
each domain to heterologous proteins that directly interact, as in the
yeast two-hybrid system,44 or by means of a
ternary complex with a bridging dimeric ligand, as illustrated in
Figure 4. Several such chemical inducers
of dimerization, including synthetic compounds and natural products
such as FK506 (which mediates the interactions of FKBP12 with
calcineurin) and rapamycin (which mediates the interaction of FKBP12
with FRB, the FKBP12-rapamycin–binding domain of FRAP), have been
examined for their ability to pharmacologically control gene
expression.45 46 For example, coexpression of
chimeric constructs consisting of a site-specific DNA-binding domain
fused to FKBP12 and the carboxy-terminal transactivation domain of
nuclear factor-
B linked to FRB will effectively
transactivate engineered target reporter genes in a
rapamycin-dependent manner,47 as schematized in
Figure 4. Since rapamycin itself is growth inhibitory and
immunosuppressive, nontoxic derivatives have been synthesized and
compensatory mutations in FRB have been identified that still allow for
ternary complex formation and transcriptional
activation.47 Although this approach has not yet
been demonstrated in vivo, the potential utility of small dimeric
ligands for regulation of transcription in intact animals is
considerable.
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Future Directions
Conditional Recombination
Substantial impetus for the development of temporally regulated
gene expression systems has come from the results of recent
gene-targeting experiments in the mouse, where loss-of-function
mutations have highlighted the potential multiplicity of functions of
individual gene products. For example, endothelin-1 is a 21–amino
acid peptide originally characterized as a potent
vasoconstrictor.48 Remarkably, homozygous mice
with targeted disruption of the endothelin-1 locus display a range of
cardiovascular malformations in pharyngeal
arch–derived tissues and organs.49 50 Similarly,
the connexin43 gene encodes a gap junction channel critical for
electrotonic coupling and impulse propagation in the heart, yet mice
lacking connexin43 develop right ventricular outflow tract
obstruction, perhaps reflecting a defect in the migration of neural
crest–derived cells during cardiac
morphogenesis.51 52 53 However, although these
loss-of-function mutations uncover unanticipated functions for various
gene products, these early lethal outcomes confound studies of gene
function during later stages of development. Moreover, since the heart
is the earliest organ to develop, there may be a bias ascribing
embryonic lethality to defective cardiogenesis even for genes with
critical functions in a variety of tissues.54
Therefore, for genes that are widely expressed or with multiple
time-dependent functions, strategies for circumscribed gene disruption,
restricted in terms of lineage or the timing of inactivation, are
required.
Already, substantial progress has been made with lineage-specific knockouts. Several groups have demonstrated the ability of Cre recombinase, expressed under the control of tissue-specific promoters, to catalyze site-specific recombination of appropriately engineered loxP-containing genomic target lineages only within the tissue of interest.55 56 57 Nonetheless, the timing of tissue-specific recombination is subject to the same limitations of all conventional transgenic experiments; ie, the onset of recombination is determined by the properties of the specific regulatory sequences controlling the recombinase. Since few promoters are active only in the mature organism, until very recently, it has been difficult to even envision techniques to specifically inactivate genes in the adult mouse. To impart temporal control on recombinase activity, several novel strategies have been explored, including delivery of Cre recombinase by viral vectors, where anatomically restricted recombination has been achieved using either replication-deficient adenoviral or HSV vectors.58 59 60 An alternative strategy has been to fuse Cre recombinase with modified ligand-binding domains of human steroid receptors, resulting in chimeric proteins with activities dependent on administration of synthetic hormone analogues.61 62 Although Cre recombinase has also been conditionally expressed using the tetracycline-regulated system, the efficiency of site-specific recombination varied substantially from tissue to tissue.63 Thus, although the relative efficiency and utility of any of these approaches remains to be determined, the ability to control precisely both the timing and location of genomic recombination promises to greatly extend the power of traditional gene-targeting strategies.
Knockins and Conditional Gene Expression
To achieve precise lineage-specific expression of heterologous
genes, strategies using homologous recombination have recently been
described whereby endogenous protein coding regions are
replaced with foreign sequences, thereby ensuring that virtually all
potential regulatory elements, even those quite distant from site of
recombination, remain intact.64 Within the
cardiovascular system, this so-called "knockin"
strategy has been used to place Cre recombinase under the
transcriptional control of the myosin light chain-2v locus, resulting
in cardiac-restricted recombination of appropriate
loxP-flanked genomic target sites.65 A
similar knockin approach could be used to express chimeric
transcription factors, such as tTA, under the transcriptional control
of an endogenous locus. Inasmuch as the intracellular
concentration of tTA and the corresponding likelihood for undesirable
transcriptional squelching will vary as a function of the
endogenous locus selected, the previously described tTA
mutants with graded transactivation potential may prove useful in this
setting.39
Finally, it is also conceivable that homologous recombination could be used to replace upstream transcriptional regulatory elements of a gene of interest with arrays of tetO sequences, thereby rendering expression of the modified locus dependent on the appropriate transcriptional activator and its ligand. This approach would allow one to conditionally regulate endogenous gene expression at the transcriptional level, a strategy that has significant theoretical benefits, including the creation of reversible null phenotypes that do not depend on dominant-negative approaches. Such a strategy has recently been described in vitro, where enhancer elements of the pancreatic elastase I gene were replaced with a tetO element, resulting in gene expression that was induced by tTA and repressible by tetracycline, while retaining cell-type specificity.66
The ability to manipulate gene expression in the intact organism with precise control is becoming less of a theoretical concept and more of an experimental reality. The marriage of conditional gene expression systems with other existing and emerging gene-targeting technologies should provide a wealth of opportunity to study gene function, physiology, and pathophysiology in vivo. These advances in mouse molecular genetics are paralleled by equally remarkable triumphs in the ability to measure functional parameters in the intact mouse. Echocardiography and magnetic resonance imaging, hemodynamics, angiography, and electrophysiology of the mouse heart have all been described in recent years.67 68 69 70 71 72 If we are wise enough to exploit these new tools in a rational manner, we should expect to learn a great deal more about the molecular determinants of function and dysfunction in the intact organism.
Selected Abbreviations and Acronyms
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Acknowledgments
This study was supported in part by grants from the National Institutes of Health, the American Heart Association, National Center and New York City Affiliate, Inc. Dr Fishman is an Established Investigator of the American Heart Association. The author would like to thank Mansoor Husain for sharing unpublished data, Bruce Conklin for helpful discussions, Richard Kitsis and Marian Meyers for carefully reviewing this manuscript, and members of his laboratory, past and present, who have contributed to work presented in this article. He apologizes to those whose work was unavoidably excluded to fit within the constraints of this format.
Received December 10, 1997; accepted January 23, 1998.
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