Published online before print June 21, 2001, doi: 10.1161/hh1301.092687
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© 2001 American Heart Association, Inc.
Molecular Medicine |
Temporally Regulated and Tissue-Specific Gene Manipulations in the Adult and Embryonic Heart Using a Tamoxifen-Inducible Cre Protein
From the Department of Pediatrics (D.S.S., S.A.W., T.R.K., K.M.T., J.D.M.), University of Cincinnati, Children’s Hospital Medical Center, Division of Molecular Cardiovascular Biology, Cincinnati, Ohio, and Amgen Institute (M.N., M.A.C., J.M.P.), Toronto, Ontario, Canada.
Correspondence to Jeffery D. Molkentin, Department of Pediatrics, Children’s Hospital Medical Center, Division of Molecular Cardiovascular Biology, 3333 Burnet Ave, Cincinnati, OH 45229-3039. E-mail jeff.molkentin{at}chmcc.org
Abstract
Abstract—The
advent of conditional and tissue-specific recombination systems in
gene-targeted or transgenic mice has permitted an assessment of single
gene function in a temporally regulated and cell-specific manner. Here
we generated transgenic mice expressing a tamoxifen-inducible Cre
recombinase protein fused to two mutant estrogen-receptor
ligand-binding domains (MerCreMer) under the control of the 
-myosin
heavy chain promoter. These transgenic mice were crossed with the
ROSA26 lacZ-flox–targeted mice
to examine Cre recombinase activity and the fidelity of the system. The
data demonstrate essentially no Cre-mediated recombination in the
embryonic, neonatal, or adult heart in the absence of inducing agent
but >80% recombination after only four tamoxifen injections.
Expression of the MerCreMer fusion protein within the adult heart did
not affect cardiac performance, cellular architecture, or
expression of hypertrophic marker genes, demonstrating that the
transgene-encoded protein is relatively innocuous. In summary,
MerCreMer transgenic mice represent a tool for temporally
regulated inactivation of any loxP-targeted gene within the developing
and adult heart or for specifically directing recombination and
expression of a loxP-inactivated cardiac transgene in the
heart.
Key Words: cardiac • cre recombinase • genetics • inducible gene expression • embryo
The use of site-specific recombinase, such as Cre or Flp, has permitted an evaluation of gene function within genetically modified animals in a tissue-specific manner.1 Typically, a gene of interest is flanked with recombinase recognition sequences (loxP sites for Cre), which permit subsequent recombination and gene disruption coincident with expression of the appropriate enzyme.1 Expression of a site-specific recombinase is achieved either through a tissue-restricted transgene or by inserting the recombinase cDNA into a genetic locus with a known tissue expression profile. Tissue-specific gene disruption is often used to circumvent embryonic or fetal lethality associated with complete somatic disruption, thus permitting an examination of gene function in the tissue of interest or at later developmental stages. However, one significant limitation of the present approach is the inability to control the timing of Cre- or Flp-mediated recombination, because gene disruption closely parallels the earliest expression profile of the chosen promoter. Examples have been reported in which Cre-mediated (tissue-specific) gene disruption still results in embryonic, fetal, or neonatal lethality, so that the adult function of a certain gene cannot be examined.2 3 4 5 6 Because the heart is the first functioning organ system within the developing embryo, a traditional tissue-specific recombination strategy could easily result in developmental lethality, excluding an assessment of gene function in the neonatal or adult heart.7 To circumvent this limitation, we have generated transgenic mice expressing a tamoxifen-inducible Cre-fusion protein specifically within the heart.
Materials and Methods
All experiments were conducted in accordance with the Guide for the Use and Care of Laboratory Animals and approved by the Institutional Animal Care and Use Committee.
A cDNA encoding a double-fusion protein between two mutant
estrogen receptor domains on either side of Cre recombinase was
subcloned as a SalI fragment
into the cardiac-specific -myosin heavy chain (MHC) 5.5-kb promoter
construct.8 9 The
MerCreMer fusion cDNA (gift of Michael Reth, Max-Plank Institute,
Freiburg, Germany) encodes the mutated murine estrogen receptor
ligand–binding domain (amino acids 281 to 599, G525R), which is
insensitive to estrogen but sensitive to
tamoxifen.9 The resulting
-MHC-MerCreMer DNA fragment was injected into pronuclei of freshly
fertilized oocytes from FVB mice to produce transgenic animals. FVB
founder transgenic mice are presently being crossed into the
C57BL/6SV129 genetic background (fourth generation at present) to
generate a strain more suitable for traditional gene-targeting
experiments. Transgene expression was verified by Western blotting of
100 µg of cardiac protein extract from 3- and 6-week-old FVB
transgenic mice using Cre-specific antisera at a dilution of 1:5000
(Covance). Western blotting and the subsequent detection procedure were
performed as previously
described.10 Similar levels
of protein expression were observed between FVB transgenic mice and
outbred C57BL/6SV129 transgenic mice (data not shown).
To induce Cre recombination, adult MerCreMer transgenic mice (3 to 12 weeks of age) were treated with tamoxifen (Sigma) by intraperitoneal injection once a day for 4 to 6 days at a dosage of 20 mg/kg per day. To examine recombination in embryos, pregnant mice were treated with tamoxifen by intraperitoneal injection once a day for 4 days at 20 mg/kg day. Tamoxifen was dissolved in 60% ethanol at a concentration of 5 mg/mL. Hearts from tamoxifen- or saline-treated control mice were removed and perfused in a retrograde manner with phosphate-buffered saline through the aorta, left ventricle, and coronary vasculature for 2 minutes followed by overnight perfusion at room temperature with X-gal staining solution to detect lacZ activity throughout the heart.11 RNA dot blotting and transthoracic echocardiography were performed as described previously.12 DNA Southern blotting to detect Cre-mediated recombination used a probe specific to an N-terminal sequence in lacZ and an EcoRI digest of genomic DNA. The lacZ Southern probe (470 base pairs) was generated by polymerase chain reaction using the following primers: lacZ up 5' GTCACACTACGTCTGAACGT-3' and lacZ down 5' CTGCACCATTCGCGTTACG-3'.
Results
The estrogen receptor is a hormone-activated transcription factor that is usually sequestered by heat-shock proteins in the absence of ligand. In the presence of 17ß-estradiol, the estrogen receptor is released, allowing participation in transcriptional regulatory complexes. Since its initial description, the ligand-binding domain of the estrogen receptor (amino acids 281 to 599) has been extensively used as a strategy for fusion protein sequestration within mammalian cells.13 More recently, a mutation in the estrogen receptor at amino acid 525 (glycine to arginine) was described that rendered it insensitive to 17ß-estradiol but sensitive to the estrogen antagonist tamoxifen.14 Use of this mutant estrogen receptor (Mer) domain in fusion protein strategies permits estrogen-insensitive but tamoxifen-inducible activity within mammalian cells.
A cDNA consisting of Cre recombinase fused to a Mer-encoding
sequence at both the N- and C-termini
(MerCreMer)9 was subcloned
into the cardiac-specific -MHC 5.5-kb promoter construct to permit
generation of transgenic mice
(Figure 1A
).8 This
double-fusion strategy was shown to more effectively reduce promiscuous
Cre activity in mammalian
cells.9 15
Interestingly, the double-fusion protein also demonstrates
substantially greater Cre recombinase activity compared with a single
CreMer fusion, suggesting that the double-fusion strategy somehow
enhances the intrinsic recombinase activity of Cre or its ability to
access DNA sites.15
|
Two stable transgenic mouse lines were generated expressing
the MerCreMer fusion protein in the heart, both of which were viable
with phenotypically normal hearts. One of the transgenic lines (line 2)
demonstrated only weak MerCreMer protein expression in the neonatal
heart, which was downregulated in adulthood (data not shown). However,
line 1 transgenic mice demonstrated robust MerCreMer protein expression
(107 kDa) in the juvenile and adult heart, as revealed with
Cre-specific antisera and Western blotting at both 3 and 6 weeks of age
(Figure 1B
).
To examine the activity of the MerCreMer protein, line 1
transgenic mice were crossed with the
ROSA26 lacZ-loxP reporter
strain as an indicator of Cre-mediated recombination in the heart. The
ROSA26 lacZ-loxP reporter
strain contains a Cre-dependent, loxP-inactivated lacZ cDNA
cassette targeted within the ubiquitously expressed
ROSA26
locus16
(Figure 2A). Cre-mediated recombination of this allele
deletes neomycin and a series of polyadenylation sequences, resulting
in the juxtaposition of a splice acceptor site and the lacZ
cDNA.5 The MerCerMer
transgene (tg/0) was bred into the ROSA26
lacZ-loxP background (lacZ/0), generating
double-heterozygous mice (MCMxROSA) for subsequent
analysis.
|
Hearts from double-heterozygous mice were harvested at
embryonic day 17 (E17), 17 days after birth, 6 weeks of age, and 12
weeks of age and subjected to X-gal staining as an indicator of
Cre-mediated recombination. Remarkably, untreated double-heterozygous
mice (MCMxROSA) showed no detectable lacZ activity in either the
ventricles or atria at E17, 17 days postnatal, or 6 weeks of age
(Figures 2B and 3A). Such a result was unexpected, because
even low levels of unregulated Cre activity result in irreversible
recombination, which has a cumulative effect on lacZ staining. Whereas
the overall lack of lacZ staining up through 6 weeks of age indicated
extremely tight regulation, we did occasionally observe a single cell
with lacZ staining in histological tissue sections
(data not shown). To more carefully examine the extent of background
recombination, significantly older (3-month-old) double-heterozygous
mice were examined. By 3 months of age, untreated double-heterozygous
mice demonstrated approximately 1% spurious recombination within the
heart as estimated from whole mount and histological
sections
(Figure 2B and data not shown). These results indicate that
the MerCreMer transgenic protein is, on the whole, tightly regulated
within the heart so that only very low levels of unregulated
recombination are observed in the absence of tamoxifen.
|
Treatment of double-heterozygous mice with tamoxifen for
only 5 days (4 separate intraperitoneal injections)
was sufficient to induce robust recombination and lacZ activity in a
uniform profile throughout the embryonic, neonatal, and adult heart
(Figure 2B). To assess tamoxifen-induced recombination in the
embryonic heart, pregnant females were treated with drug from E12 to
E16, the embryos were harvested at E17, and the hearts were removed for
lacZ staining. Although the -MHC promoter drives high levels of
expression in the postnatal and adult heart, significant expression is
also observed in the embryonic
heart.17 Cre-mediated
recombination was observed in utero, indicating that the MerCreMer
transgene can be used to manipulate gene expression in the embryonic
heart
(Figure 2B). Hearts harvested on E17 showed somewhat
less-robust lacZ staining when viewed in whole mount, attributable
largely to the staining procedure. Given their size, embryonic hearts
were simply emersed in staining solution, whereas neonatal and adult
hearts were slowly perfused with staining solution through the
coronary vasculature, exposing essentially all
cardiomyocytes. Despite the differing staining procedures,
histological sectioning of E17 hearts revealed a robust
and uniform profile of lacZ expression in the outer few cell layers
exposed to the staining solution (data not shown). Finally, 3-month-old
double-heterozygous mice also demonstrated robust lacZ
expression after 5 days of tamoxifen treatment
(Figure 2B). Collectively, these data demonstrate efficient
Cre-mediated recombination in the embryonic, neonatal, and adult mouse
heart.
To more carefully evaluate the characteristics of the
MerCreMer transgene and tamoxifen-induced recombination, large cohorts
of mice were examined at 6 weeks of age. Six-week-old
double-heterozygous mice treated with tamoxifen demonstrated lacZ
staining throughout both right and left ventricles and atria, although
atrial staining appeared less robust because of its thinness
(Figure 3A). Within the ventricles of 6-week-old mice,
staining was homogenous throughout the myocardial, endocardial, and
epicardial cell layers
(Figure 3B). Untreated double-transgenic mice also displayed
a homogenous profile of nearly absent lacZ staining in the myocardial,
endocardial, and epicardial cell layers at 6 weeks of age
(Figures 3A through 3C). Identical results were observed in 6
independent experiments. Additional controls showed no lacZ staining in
single heterozygous ROSA26
lacZ-loxP mice or in single heterozygous MerCreMer
transgenic mice treated with tamoxifen
(Figure 3A). No difference was observed in levels of
unregulated or inducible lacZ staining between male and female mice at
6 weeks of age (data not shown).
At 6 weeks of age, lacZ-stained hearts were
histologically sectioned to more carefully evaluate the
extent of recombination after 5 days of tamoxifen treatment
(Figures 3B and 3C). Nearly all of the myocytes demonstrated
lacZ activity in double-heterozygous mice (MCMxROSA) treated with
tamoxifen, whereas untreated mice showed undetectable or extremely low
levels of lacZ staining
(Figures 3B and 3C). Qualitative assessment of
histological sections suggested a recombination
frequency of >80% after 5 days of tamoxifen administration in 5- to
6-week-old mice. To additionally evaluate the extent of Cre-mediated
recombination, Southern blotting was performed with a probe specific to
the ROSA 26 locus
(neomycin-lacZ cassette). Untreated double-heterozygote mice showed no
recombination at baseline at 6 weeks of age (lane 2), whereas 5 days of
tamoxifen treatment revealed >70% recombination in the heart (lane 1)
(Figure 3D). However, given that the heart contains a fair
number of nonmyocytes (fibroblasts, infiltrates, and
endothelial cells) that do not express the MerCreMer
protein, the myocyte-specific recombination frequency is undoubtedly
>70% as measured at 6 weeks of age. No recombination was observed in
the brain, kidney, lung, liver, and skeletal muscle of
double-heterozygous mice treated with tamoxifen
(Figure 3D). Collectively, these data demonstrate efficient
tamoxifen-induced Cre-mediated recombination specifically in the heart
of MerCreMer transgenic mice. More importantly, the Southern blot data
also confirm the histological data, demonstrating
nearly undetectable levels of background recombination at 6 weeks of
age.
MerCreMer transgenic mice represent a valuable
genetic tool for inducing the inactivation of any loxP-targeted gene
within the heart. However, for such a reagent to be useful, the
MerCreMer protein itself should not adversely affect the heart.
Accordingly, echocardiography was performed in
8-week-old MerCreMer transgenic mice (± tamoxifen for 5 days) and
compared with nontransgenic littermate controls. The results show no
alterations in cardiac chamber dimensions, wall thicknesses, or
fractional shortening in MerCreMer transgene either with or without
tamoxifen treatment
(Table).
It should be noted that MerCreMer transgenic mice were allowed to
recover from tamoxifen treatment for 6 days before
echocardiography to eliminate any potential
nonspecific effects associated with momentary tamoxifen
administration.
|
Heart weights of MerCreMer transgenic mice were similar to
nontransgenic littermate control hearts at 8 weeks of age, and H&E- and
trichrome-stained histological sections failed to
identify any pathology
(Figure 4A). More importantly, tamoxifen-treated MerCreMer
transgenic mice (6 days after treatment) did not show upregulated
expression of hypertrophy marker genes compared with
wild-type control mice
(Figure 4B). In contrast, control MEK1 transgenic mice with
demonstrable hypertrophy showed significant upregulated
expression of atrial natriuretic factor, brain
natriuretic peptide, skeletal -actin, and ß-MHC mRNA
(Figure 4B).12
Collectively, these data demonstrate that the MerCreMer transgene is
relatively innocuous to the heart.
|
Discussion
The ability to control tissue specificity of gene
deletions in the mouse using Cre-lox technology has profoundly advanced
mouse genetics and the ability to examine single gene function in vivo.
However, one significant limitation of the traditional Cre-lox approach
is an inability to temporally control genetic recombination. Typically,
a loxP-targeted gene undergoes Cre-mediated recombination coincident
with Cre expression directed by a promoter region of interest. In this
study, we describe transgenic mice that express a modified Cre
recombinase fusion protein in the heart under the transcriptional
control of the -MHC promoter. Whereas transgene expression from this
promoter is largely constitutive throughout development and in the
adult heart, the Cre recombinase fusion protein is
inactivated by two mutant estrogen receptor ligand-binding
domains. Such an approach only requires a single transgene to achieve
cardiac-specific and temporally regulated recombination in vivo.
Alternative cardiac-specific gene regulatory strategies, such as the
tetracycline-responsive activator and transcriptional
regulatory region, have also been recently
described.18 However, the
tetracycline-inducible system typically requires two independent
transgenes (binary), one expressing the
tet-activator/repressor in the tissue of interest
and one containing a chimeric tetracycline-regulated promoter fused to
a cDNA encoding Cre recombinase. Although such a tetracycline-inducible
system for regulating Cre-mediated recombination in the heart has yet
to be described, it remains entirely feasible, because the proof of
concept has already been
demonstrated.19 20 21
However, a binary tetracycline-inducible approach requires a more
complicated breeding strategy compared with the monogenic MerCreMer
approach described herein. In addition, the potential fidelity
(leakiness) of a tetracycline-regulated Cre recombinase system is
uncertain.
While this manuscript was in review, Minamino et
al22 described the
generation and characterization of a RU486-regulated Cre protein in the
heart. These investigators used a construct in which Cre was fused to
the ligand-binding domain of the human progesterone receptor under the
control of the -MHC promoter. Similar to our study, Minamino et
al22 demonstrated
drug-regulated Cre recombination in a temporally controlled manner in
the mouse heart. However, the approach used by Minamino et al resulted
in slightly greater background recombination compared with the
tamoxifen-inducible MerCreMer approach described here
(Figures 2 and 3). It is likely that our approach using both
an N- and C-termini Mer domain imparts tighter regulation compared with
the single progesterone receptor domain fusion used by Minamino et
al.22 Indeed, the double Mer
fusion protein (MerCreMer) was previously shown to substantially reduce
promiscuous Cre activity in cultured cells and even to enhance
Cre-mediated recombination compared with a single CreMer fusion
protein.9 15
Another difference between the tamoxifen- and
RU486-inducible systems relates to the ability to perform developmental
gene manipulations in embryos. RU486, an antiprogesterone, can be more
detrimental to early-stage embryos compared with the antiestrogen
compound tamoxifen.23
Indeed, a tamoxifen-regulated CreMer transgene expressed in the
developing neural tube has already been shown to promote regulated gene
recombination in early- and mid-gestation mouse
embryos.24 Similarly, we
show that tamoxifen administration to pregnant mice between E12 and E16
promoted recombination in the embryonic heart
(Figure 2B). Whereas the -MHC promoter is widely used for
its characteristic expression pattern in the neonatal and adult heart,
embryonic expression is also
significant.17 A similar yet
slightly lower dosage of tamoxifen (7.5 mg/kg per day) administered
daily between E12 and E18 in pregnant CD-1 mice did not compromise
development or induce abortion, although endometrial hyperplasia and
polyploid adenomas were identified in 30% to 50% of female offspring
at 
1 year of age.25
Despite these concerns, the results of the present study indicate
that MerCreMer transgenic mice can be used to temporally regulate gene
manipulations within the developing mouse heart without causing
embryonic lethality.
Here we measured Cre-mediated recombination within the ROSA26 gene locus, which is ubiquitously expressed and constitutively active in the mouse. Given its active confirmation with respect to chromatin, it is formally possible that the ROSA26 locus is more permissive to site-specific recombination compared with other genetic loci. A final technical consideration relates to the use of tamoxifen as an inducing agent. Although echocardiographic assessment of tamoxifen-treated MerCreMer transgenic mice did not reveal obvious defects in cardiac chamber dimensions 6 days after drug treatment, nor were hypertrophic marker genes significantly activated, it remains possible that tamoxifen might alter some aspect of cardiac physiology or biochemistry. However, decreasing the dose of tamoxifen (10 mg/kg per day) or the time course of treatment might resolve any potential alterations. Indeed, we have observed that even a single tamoxifen injection in 3-week-old double-heterozygous mice induced significant lacZ expression (data not shown).
The most obvious application of the MerCreMer transgenic
line is to promote temporally regulated deletion of loxP-targeted genes
in vivo. However, the MerCreMer transgene can also be used to
temporally regulate expression of another unrelated transgene within
the heart. Such a transgene would be constructed such that a loxP
flanked stuffer sequence is placed immediately upstream of a given
cDNA, rendering it inactive. Cre-mediated excision of such a stuffer
region would result in the juxtaposition of the downstream cDNA and the
given promoter, as shown in
Figure 2A. In this manner, a transgene could be specifically
activated in the adult heart, bypassing developmental affects
associated with unregulated transgenesis. In summary, MerCreMer
transgenic mice will permit temporally regulated activation or
inactivation of a properly designed transgene or a loxP-targeted
genetic locus within the heart. Such applications will undoubtedly
expand our ability to genetically dissect the function of putative
disease-causing genes within either the developing or adult
heart.
Acknowledgments
This work was supported by the National Institutes of Health and a Pew charitable trust scholar award (to J.D.M.).
Footnotes
Original received February 2, 2001; revision received May 8, 2001; accepted May 8, 2001.
References
1. Fiering S, Bender MA, Groudine M. Analysis of mammalian cis-regulatory DNA elements by homologous recombination. Methods Enzymol. 1999;306:42–66.
2. Shafi R, Iyer SP, Ellies LG, O’Donnell N, Marek KW, Chui D, Hart GW, Marth JD. The O-GlcNAc transferase gene resides on the X chromosome and is essential for embryonic stem cell viability and mouse ontogeny. Proc Natl Acad Sci U S A. 2000;97:5735–5739.
3. Li H, Wang J, Wilhelmsson H, Hansson A, Thoren P, Duffy J, Rustin P, Larsson NG. Genetic modification of survival in tissue-specific knockout mice with mitochondrial cardiomyopathy. Proc Natl Acad Sci U S A. 2000;97:3467–3472.
4. Haigh JJ, Gerber HP, Ferrara N, Wagner EF. Conditional inactivation of VEGF-A in areas of collagen2a1 expression results in embryonic lethality in the heterozygous state. Development. 2000;127:1445–1453.
5. Nozaki M, Ohishi K, Yamada N, Kinoshita T, Nagy A, Takeda J. Developmental abnormalities of glycosylphosphatidylinositol-anchor-deficient embryos revealed by Cre/loxP system. Lab Invest. 1999;79:293–299.
6. Liu JL, Grinberg A, Westphal H, Sauer B, Accili D, Karas M, LeRoith D. Insulin-like growth factor-I affects perinatal lethality and postnatal development in a gene dosage-dependent manner: manipulation using the Cre/loxP system in transgenic mice. Mol Endocrinol. 1998;12:1452–1462.
7. Copp AJ. Death before birth: clues from gene knockouts and mutations. Trends Genet. 1995;11:87–93.
8. Palermo J, Gulick J, Colbert M, Fewell J, Robbins J. Transgenic remodeling of the contractile apparatus in the mammalian heart. Circ Res. 1996;78:504–509.
9. Zhang Y, Riesterer C, Ayrall AM, Sablitzky F, Littlewood TD, Reth M. Inducible site-directed recombination in mouse embryonic stem cells. Nucleic Acids Res. 1996;24:543–548.
10. De Windt LJ, Lim HW, Haq S, Force T, Molkentin JD. Calcineurin promotes protein kinase C and c-Jun NH2-terminal kinase activation in the heart: cross-talk between cardiac hypertrophic signaling pathways. J Biol Chem. 2000;275:13571–13579.
11. Cheng TC, Hanley TA, Mudd J, Merlie JP, Olson EN. Mapping of myogenin transcription during embryogenesis using transgenes linked to the myogenin control region. J Cell Biol. 1992;119:1649–1656.
12. Bueno OF, De Windt LJ, Tymitz KM, Witt SA, Kimball TR, Klevitsky R, Hewett TE, Jones SP, Lefer DJ, Peng CF, Kitsis RN, Molkentin JD. The MEK1-ERK1/2 signaling pathway promotes compensated cardiac hypertrophy in transgenic mice. EMBO J. 2000;19:6341–6350.
13. Eilers M, Picard D, Yamamoto KR, Bishop JM. Chimaeras of myc oncoprotein and steroid receptors cause hormone-dependent transformation of cells. Nature. 1989;340:66–68.
14. Littlewood TD, Hancock DC, Danielian PS, Parker MG, Evan GI. A modified oestrogen receptor ligand-binding domain as an improved switch for the regulation of heterologous proteins. Nucleic Acids Res. 1995;23:1686–1690.
15. Verrou C, Zhang Y, Zurn C, Schamel WW, Reth M. Comparison of the tamoxifen regulated chimeric Cre recombinases MerCreMer and CreMer. Biol Chem. 1999;380:1435–1438.
16. Soriano P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet. 1999;21:70–71.
17. Luo Y, Ferreira-Cornwell M, Baldwin H, Kostetskii I, Lenox J, Lieberman M, Radice G. Rescuing the N-cadherin knockout by cardiac-specific expression of N- or E-cadherin. Development. 2001;128:459–469.
18. Fishman GI. Timing is everything in life: conditional transgene expression in the cardiovascular system. Circ Res. 1998;82:837–844.
19. Yu Z, Redfern CS, Fishman GI. Conditional transgene expression in the heart. Circ Res. 1996;79:691–697.
20. Bowman JC, Steinberg SF, Jiang T, Geenen DL, Fishman GI, Buttrick PM. Expression of protein kinase C beta in the heart causes hypertrophy in adult mice and sudden death in neonates. J Clin Invest. 1997;100:2189–2195.
21. Redfern CH, Degtyarev MY, Kwa AT, Salomonis N, Cotte N, Nanevicz T, Fidelman N, Desai K, Vranizan K, Lee EK, Coward P, Shah N, Warrington JA, Fishman GI, Bernstein D, Baker AJ, Conklin BR. Conditional expression of a Gi-coupled receptor causes ventricular conduction delay and a lethal cardiomyopathy. Proc Natl Acad Sci U S A. 2000;97:4826–4831.
22. Minamino T, Gaussin V, DeMayo FJ, Schneider MD. Inducible gene targeting in postnatal myocardium by cardiac-specific expression of a hormone-activated Cre fusion protein. Circ Res. 2001;88:587–592.
23. Sadek S, Bell SC. The effects of the antihormones RU486 and tamoxifen on fetoplacental development and placental bed vascularisation in the rat: a model for intrauterine fetal growth retardation. Br J Obstet Gynaecol. 1996;103:630–641.
24. Danielian PS, Muccino D, Rowitch DH, Michael SK, McMahon AP. Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase. Curr Biol. 1998;8:1323–1326.
25. Diwan BA, Anderson LM, Ward JM. Proliferative lesions of oviduct and uterus in CD-1 mice exposed prenatally to tamoxifen. Carcinogenesis. 1997;18:2009–2014.
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 |
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 D. Catalucci, D.-H. Zhang, J. DeSantiago, F. Aimond, G. Barbara, J. Chemin, D. Bonci, E. Picht, F. Rusconi, N. D. Dalton, et al. Akt regulates L-type Ca2+ channel activity by modulating Cav{alpha}1 protein stability J. Cell Biol., March 23, 2009; 184(6): 923 - 933. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Zheng, H. Cheng, X. Li, J. Zhang, L. Cui, K. Ouyang, L. Han, T. Zhao, Y. Gu, N. D. Dalton, et al. Cardiac-specific ablation of Cypher leads to a severe form of dilated cardiomyopathy with premature death Hum. Mol. Genet., February 15, 2009; 18(4): 701 - 713. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. C. Zelarayan, C. Noack, B. Sekkali, J. Kmecova, C. Gehrke, A. Renger, M.-P. Zafiriou, R. van der Nagel, R. Dietz, L. J. de Windt, et al. {beta}-Catenin downregulation attenuates ischemic cardiac remodeling through enhanced resident precursor cell differentiation PNAS, December 16, 2008; 105(50): 19762 - 19767. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. A. da Costa Martins, M. Bourajjaj, M. Gladka, M. Kortland, R. J. van Oort, Y. M. Pinto, J. D. Molkentin, and L. J. De Windt Conditional Dicer Gene Deletion in the Postnatal Myocardium Provokes Spontaneous Cardiac Remodeling Circulation, October 7, 2008; 118(15): 1567 - 1576. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. E. Briggs, M. Takeda, A. E. Cuadra, H. Wakimoto, M. H. Marks, A. J. Walker, T. Seki, S. P. Oh, J. T. Lu, C. Sumners, et al. Perinatal Loss of Nkx2-5 Results in Rapid Conduction and Contraction Defects Circ. Res., September 12, 2008; 103(6): 580 - 590. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. W. Raake, L. E. Vinge, E. Gao, M. Boucher, G. Rengo, X. Chen, B. R. DeGeorge Jr, S. Matkovich, S. R. Houser, P. Most, et al. G Protein-Coupled Receptor Kinase 2 Ablation in Cardiac Myocytes Before or After Myocardial Infarction Prevents Heart Failure Circ. Res., August 15, 2008; 103(4): 413 - 422. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Yoshioka, K. Imahashi, S. A. Gabel, W. A. Chutkow, A. A. Burds, J. Gannon, P. C. Schulze, C. MacGillivray, R. E. London, E. Murphy, et al. Targeted Deletion of Thioredoxin-Interacting Protein Regulates Cardiac Dysfunction in Response to Pressure Overload Circ. Res., December 7, 2007; 101(12): 1328 - 1338. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Wang Mitogen-Activated Protein Kinases in Heart Development and Diseases Circulation, September 18, 2007; 116(12): 1413 - 1423. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Ruan, S. Mitchell, M. Vainoriene, Q. Lou, L.-H. Xie, S. Ren, J. I. Goldhaber, and Y. Wang Gi{alpha}1-Mediated Cardiac Electrophysiological Remodeling and Arrhythmia in Hypertrophic Cardiomyopathy Circulation, August 7, 2007; 116(6): 596 - 605. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Xiao, Y. Sun, J.-H. Ding, S. Lin, D. W. Rose, M. G. Rosenfeld, X.-D. Fu, and X. Li Splicing Regulator SC35 Is Essential for Genomic Stability and Cell Proliferation during Mammalian Organogenesis Mol. Cell. Biol., August 1, 2007; 27(15): 5393 - 5402. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. E. Yutzey and J. Robbins Principles of Genetic Murine Models for Cardiac Disease Circulation, February 13, 2007; 115(6): 792 - 799. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Peng, K. Raddatz, J. D. Molkentin, Y. Wu, S. Labeit, H. Granzier, and M. Gotthardt Cardiac Hypertrophy and Reduced Contractility in Hearts Deficient in the Titin Kinase Region Circulation, February 13, 2007; 115(6): 743 - 751. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Xiong, T. Yajima, B.-K. Lim, A. Stenbit, A. Dublin, N. D. Dalton, D. Summers-Torres, J. D. Molkentin, H. Duplain, R. Wessely, et al. Inducible Cardiac-Restricted Expression of Enteroviral Protease 2A Is Sufficient to Induce Dilated Cardiomyopathy Circulation, January 2, 2007; 115(1): 94 - 102. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Zhou, J. Qu, X. P. Yi, K. Graber, L. Huber, X. Wang, A. M. Gerdes, and F. Li Upregulation of {gamma}-catenin compensates for the loss of beta-catenin in adult cardiomyocytes Am J Physiol Heart Circ Physiol, January 1, 2007; 292(1): H270 - H276. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H.-L. Noh, K. Okajima, J. D. Molkentin, S. Homma, and I. J. Goldberg Acute lipoprotein lipase deletion in adult mice leads to dyslipidemia and cardiac dysfunction Am J Physiol Endocrinol Metab, October 1, 2006; 291(4): E755 - E760. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Chen, S. P. Shevtsov, E. Hsich, L. Cui, S. Haq, M. Aronovitz, R. Kerkela, J. D. Molkentin, R. Liao, R. N. Salomon, et al. The {beta}-Catenin/T-Cell Factor/Lymphocyte Enhancer Factor Signaling Pathway Is Required for Normal and Stress-Induced Cardiac Hypertrophy Mol. Cell. Biol., June 15, 2006; 26(12): 4462 - 4473. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. J. Lavine, A. C. White, C. Park, C. S. Smith, K. Choi, F. Long, C.-c. Hui, and D. M. Ornitz Fibroblast growth factor signals regulate a wave of Hedgehog activation that is essential for coronary vascular development. Genes & Dev., June 15, 2006; 20(12): 1651 - 1666. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. J. Martindale, R. Fernandez, D. Thuerauf, R. Whittaker, N. Gude, M. A. Sussman, and C. C. Glembotski Endoplasmic Reticulum Stress Gene Induction and Protection From Ischemia/Reperfusion Injury in the Hearts of Transgenic Mice With a Tamoxifen-Regulated Form of ATF6 Circ. Res., May 12, 2006; 98(9): 1186 - 1193. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Satoh, H. Ogita, K. Takeshita, Y. Mukai, D. J. Kwiatkowski, and J. K. Liao Requirement of Rac1 in the development of cardiac hypertrophy PNAS, May 9, 2006; 103(19): 7432 - 7437. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Mitchell, A. Ota, W. Foster, B. Zhang, Z. Fang, S. Patel, S. F. Nelson, S. Horvath, and Y. Wang Distinct gene expression profiles in adult mouse heart following targeted MAP kinase activation Physiol Genomics, March 13, 2006; 25(1): 50 - 59. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Fan, Y.-P. Jiang, Z. Lu, D. W. Martin, D. J. Kelly, J. M. Zuckerman, L. M. Ballou, I. S. Cohen, and R. Z. Lin A Transgenic Mouse Model of Heart Failure Using Inducible G{alpha}q J. Biol. Chem., December 2, 2005; 280(48): 40337 - 40346. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Parlakian, C. Charvet, B. Escoubet, M. Mericskay, J. D. Molkentin, G. Gary-Bobo, L. J. De Windt, M.-A. Ludosky, D. Paulin, D. Daegelen, et al. Temporally Controlled Onset of Dilated Cardiomyopathy Through Disruption of the SRF Gene in Adult Heart Circulation, November 8, 2005; 112(19): 2930 - 2939. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Li, V. V. Patel, I. Kostetskii, Y. Xiong, A. F. Chu, J. T. Jacobson, C. Yu, G. E. Morley, J. D. Molkentin, and G. L. Radice Cardiac-Specific Loss of N-Cadherin Leads to Alteration in Connexins With Conduction Slowing and Arrhythmogenesis Circ. Res., September 2, 2005; 97(5): 474 - 481. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Kostetskii, J. Li, Y. Xiong, R. Zhou, V. A. Ferrari, V. V. Patel, J. D. Molkentin, and G. L. Radice Induced Deletion of the N-Cadherin Gene in the Heart Leads to Dissolution of the Intercalated Disc Structure Circ. Res., February 18, 2005; 96(3): 346 - 354. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Syed, A. Odley, H. S. Hahn, E. W. Brunskill, R. A. Lynch, Y. Marreez, A. Sanbe, J. Robbins, and G. W. Dorn II Physiological Growth Synergizes With Pathological Genes in Experimental Cardiomyopathy Circ. Res., December 10, 2004; 95(12): 1200 - 1206. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Conrad, C. Jakupoglu, S. G. Moreno, S. Lippl, A. Banjac, M. Schneider, H. Beck, A. K. Hatzopoulos, U. Just, F. Sinowatz, et al. Essential Role for Mitochondrial Thioredoxin Reductase in Hematopoiesis, Heart Development, and Heart Function Mol. Cell. Biol., November 1, 2004; 24(21): 9414 - 9423. [Abstract] [Full Text] [PDF]
|
|
|
|
|
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K. Matsuura, H. Wada, T. Nagai, Y. Iijima, T. Minamino, M. Sano, H. Akazawa, J. D. Molkentin, H. Kasanuki, and I. Komuro Cardiomyocytes fuse with surrounding noncardiomyocytes and reenter the cell cycle J. Cell Biol., October 25, 2004; 167(2): 351 - 363. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Xu, M. Morishima, J. N. Wylie, R. J. Schwartz, B. G. Bruneau, E. A. Lindsay, and A. Baldini Tbx1 has a dual role in the morphogenesis of the cardiac outflow tract Development, July 1, 2004; 131(13): 3217 - 3227. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Parlakian, D. Tuil, G. Hamard, G. Tavernier, D. Hentzen, J.-P. Concordet, D. Paulin, Z. Li, and D. Daegelen Targeted Inactivation of Serum Response Factor in the Developing Heart Results in Myocardial Defects and Embryonic Lethality Mol. Cell. Biol., June 15, 2004; 24(12): 5281 - 5289. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Sanbe, J. Gulick, M. C. Hanks, Q. Liang, H. Osinska, and J. Robbins Reengineering Inducible Cardiac-Specific Transgenesis With an Attenuated Myosin Heavy Chain Promoter Circ. Res., April 4, 2003; 92(6): 609 - 616. [Abstract] [Full Text] [PDF]
|
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|
|
 |
|
 B. J Wilkins and J. D Molkentin Calcineurin and cardiac hypertrophy: Where have we been? Where are we going? J. Physiol., May 15, 2002; 541(1): 1 - 8. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B.G. PETRICH, P. LIAO, and Y. WANG Using a Gene-switch Transgenic Approach to Dissect Distinct Roles of MAP Kinases in Heart Failure Cold Spring Harb Symp Quant Biol, January 1, 2002; 67(0): 429 - 438. [Abstract] [PDF]
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