© 1996 American Heart Association, Inc.
Articles |
Conditional Transgene Expression in the Heart
the Section of Molecular Cardiology, Albert Einstein College of Medicine, Bronx, NY (Z.Y., G.I.F.), and Gladstone Institute of Cardiovascular Disease, University of California, San Francisco (C.S.R.).
Correspondence to Glenn I. Fishman, MD, Section of Molecular Cardiology, Albert Einstein College of Medicine, 1300 Morris Park Ave, Bronx, NY 10461. E-mail fishman@aecom.yu.edu.
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Abstract |
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Conditional transgene expression is a potentially useful approach to investigate complex biological systems in vivo. We recently demonstrated that tetracycline-responsive promoters could be employed to achieve regulated, cardiac-specific expression of target genes in transgenic mice. To more fully define the quantitative and spatial parameters associated with tetracycline-regulated gene expression in the heart, we crossed transgenic mice harboring either a firefly luciferase or a nuclear-localized bacterial lacZ target gene with strains expressing a tetracycline-controlled transactivator (tTA) under the regulatory control of 2.9 kb of 5' flanking sequence from the rat
-myosin heavy chain gene. Luciferase activity was induced nearly 300-fold in the hearts of binary-transgenic mice compared with mice carrying only the luciferase reporter gene. No significant transactivation was observed in any other tissues examined. Binary transgenics harboring the lacZ reporter gene showed substantial ß-galactosidase activity throughout the heart, but the response of individual cardiac myocytes was heterogeneous. For both reporter genes, tetracycline treatment fully repressed tTA-dependent transactivation. These data provide important insights into the nature of studies that can be successfully addressed using the tetracycline-regulated gene expression system in the heart.
Key Words: conditional • binary • tetracycline • transgenic • heart
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Introduction |
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Transgenesis is a powerful approach to examine gene function in the intact animal.1 2 3 By ectopically expressing heterologous genes in vivo, novel organisms can be generated with well-defined perturbations in gene expression. Moreover, mechanistic relationships may be identified, since the resulting phenotype can be attributed, either directly or secondarily, to the primary genetic alteration. Although widely applicable to many biological questions, transgenic technology is a particularly attractive tool for studies of the heart, since many complex variables interact to influence cardiovascular development and function, and these interactions may be inadequately modeled in vitro.4 5 6 7 8 9
Typically, expression of transgenes is controlled by ubiquitously expressed promoters or targeted to selected lineages by using tissue-specific regulatory elements. Several promoters that direct cardiac-specific expression of heterologous sequences have been well characterized and have been exceedingly useful for diverse cardiovascular studies.10 11 12 13 14 15 16 On the basis of the results of recent gene-targeting experiments, in addition to spatial specificity, the temporal profile of gene expression has become increasingly recognized as a critical determinant of phenotype. For example, loss-of-function mutations of individual myogenic determination factor genes, such as MyoD, Myf5, and myogenin, each lead to distinctly different developmental perturbations.17 18 19 20 21 22 Similarly, mutations of the related mouse Engrailed genes En-1 and En-2 produce unique disturbances of neural development. Yet in both instances, gene replacement studies (myogenin into the Myf5 locus, or En-2 into the En-1 locus) have demonstrated that these different outcomes reflect unique profiles of transcriptional activation rather than distinct biochemical activities.23 24 Thus, related gene products may exhibit unique properties by virtue of their patterns of expression rather than their downstream targets.
These results suggest that many important biological questions could be examined most effectively by using systems in which the timing as well as the spatial patterns of gene expression can be controlled. To improve the capacity to regulate the timing of transgene expression, a number of conditional gene expression systems have been described, primarily those based on inducible promoters such as the mouse mammary tumor virus long terminal repeat, binary strategies using nonmammalian transactivators and operators, and more recently, ligand-dependent regulatory systems derived from Escherichia coli.25 26 27 28 Although many of these systems have been highly functional in vitro, in practice their adaptation for transgenic use has been relatively disappointing and none have been widely adopted.
Recently, a regulatory system based on the tetracycline-resistance operon of E coli was described that was highly functional in vitro.29 We and others have adapted this system to achieve conditional gene expression in transgenic mice.30 31 32 Nonetheless, questions as to the extent and stability of target gene expression, as well as the overall utility of this system, remain unanswered. To more fully develop this system as a useful approach for conditional gene expression in the heart, we have now generated novel transgenic mice that specifically and stably express the tetracycline-controlled transactivator (tTA) exclusively in cardiac myocytes. We crossed these mice with strains carrying tTA-responsive target genes encoding either firefly luciferase or nuclear-localized bacterial ß-galactosidase.31 The magnitude and location of target gene expression and the degree of tetracycline-dependent repression were examined in progeny. Our results suggest that conditional gene expression using tetracyline-responsive promoters is a powerful system to manipulate gene expression in vivo and a highly useful strategy for transgenic studies of the heart.
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Materials and Methods |
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Generation of Transgenes
The cardiac-specific 2.9
tTA transgenic construct was prepared by modifying the previously described MHC-tTA plasmid30 33 to include additional 5' regulatory sequences. A 0.8-kb base pair (bp) fragment encompassing nucleotides -634 to +34 (relative to transcriptional initiation) as well as the heterologous splice donor site from MHC-tTA was excised and replaced with a 3.3-kb HindIII fragment from the rat MHC gene. The new promoter fragment extended from nucleotides -2936 to +427 within intron 1, which was joined to the existing splice acceptor site in the original plasmid. The remaining parts of the construct, including the tTA coding region and human growth hormone 3' transcriptional termination signals, were left unchanged. The tTA-dependent pUHC13-3 luciferase gene and the pUHG16-3 nuclear-localized lacZ reporter gene place the respective protein coding regions downstream of the tetracycline-responsive promoter.31 All three constructs are shown schematically in Fig 1
.
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Transgenic Mouse Production
The 2.9tTA transcription unit was separated from vector sequences by restriction enzyme digestion and agarose gel electrophoresis, then purified and equilibrated with injection buffer (10 mmol/L Tris, pH 7.4, 0.1 mmol/L EDTA), as previously described.30 Pronuclear injection was carried out in the Albert Einstein College of Medicine Transgenic Mouse Facility according to standard techniques into a B6/CBAF1 background. Founder mice were identified by Southern blot analysis of genomic DNA prepared from tail biopsies. Generation of the LU5 luciferase and G2 lacZ reporter gene strains has been described previously.31
Hybridization Analysis
Southern blots were carried out with 10 µg of genomic DNA prepared from tail biopsy samples, according to established techniques. Northern blots were performed with 
10 µg of total cardiac RNA prepared from neonatal hearts, using Trizol reagent (GIBCO-BRL). The tTA transgene or transcripts were identified by using a radiolabeled 1-kb EcoRI-BamHI coding-region fragment from plasmid pUHD15-1.29 The luciferase gene was detected by using a 2.2-kb Xho I-EcoNI from plasmid pUHC13-3,29 and the lacZ gene was detected with a 1-kb Xba I-Cla I fragment from pUHG16-3.31
Polymerase Chain Reaction (PCR) Analysis
Additional genotyping was carried out by PCR. The tTA transgene was identified with primers from the transcriptional initiation site of the rat MHC gene (5'-TCAGACCGAGATTTCTCCATCCC-3') and the splice acceptor site (5'-GAATTCAGGCTCGCCTGCAGTTGG-3'), resulting in a 535-bp PCR product. The luciferase reporter gene was identified with primers from the protein coding regions (5'-ATCCTCTAGAGGATGGAAC-3' and 5'-CGATCAAAGGACTCTGGTA-3'), producing a 524-bp product. The lacZ transgene was detected with primers from the CMV minimal promoter (5'-GGCGTGTACGGTGGGAGG-3') and the ß-galactosidase nuclear-localization signal (5'-GTTGGGAAGGGCGATCGG-3'), resulting in a 269-bp product. PCR reactions were fractioned on 1% agarose gels and the amplified bands visualized by ethidium bromide staining (see Fig 1B
).
Antibiotic Treatment
To repress tTA-dependent transactivation, animals were treated with drinking water supplemented with 2% sucrose and either tetracycline hydrochloride (1 mg/mL) or doxycycline (0.2 mg/mL) (Sigma Chemical Company). For analysis of neonates, mothers were treated with the antibiotic throughout gestation until the time of tissue harvest. Adult mice were treated for at least 7 days before analysis.
Luciferase Assay
Luciferase assays were performed using a commercially available kit (Promega), with slight modifications. Individual tissues were rapidly excised and homogenized at full speed for a total of 30 seconds (Polytron, Brinkmann) in 300 µL of ice-cold buffer I (containing 50 mmol/L K3PO4, 1 mmol/L EDTA, 1 mmol/L DTT, and 10% glycerol). The samples were then clarified in a tabletop microfuge at 4°C at 3000 rpm for 15 minutes. A portion (200 µL) of each supernatant was then transferred to a new tube and mixed with 40 µL of 5x cell culture lysis reagent. The luciferase activity in duplicate 20-µL samples was determined by using a Monolight 2010 luminometer (Analytical Luminescence Laboratory). The total cellular protein concentration in each sample was also determined, using a commercial assay kit (Bio-Rad). To determine the absolute level of luciferase protein contained in each sample, standard curves using commercially available luciferase (Sigma) were generated in parallel. Serial dilutions of pure luciferase were prepared in either buffer I or in nontransgenic cardiac lysates, with no significant difference in luciferase activity (not shown).
ß-Galactosidase Histochemistry
Expression of the lacZ reporter gene was detected histochemically. Hearts were excised, washed twice in PBS, fixed for 1 hour in 2% paraformaldehyde and 0.02% glutaraldehyde in PBS, and then rinsed again in PBS. The hearts were then incubated overnight at 30°C in a solution containing 0.1% X-gal, 2 mmol/L MgCl2, 5 mmol/L EGTA, 0.02% Nonidet P-40, 5 mmol/L K3Fe(CN)6, and 5 mmol/L K4Fe(CN)66H2O. The hearts were then embedded in paraffin, sectioned, and lightly counterstained with eosin. For some samples, heart were stained with X-gal reagent after sectioning.
Statistical Analysis
A Box-Cox transformation was performed on the level of luciferase activities to form a more nearly normal distribution. Cardiac samples were initially assigned to five groups: nontransgenic (N), 2.9tTA only (T), LU5 only (L), double transgenic (D), and double transgenic treated with tetracycline (D+). A one-way ANOVA model was used to determine whether there were significant differences in the level of luciferase expression among the five groups. Differences between individual groups were analyzed post hoc using Duncan's multiple range test. For noncardiac samples, values from single- and double-transgenic mice were compared by permutation tests. In all cases, a value of P<.05 was considered significant. All results are presented graphically as mean±SEM.
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Results |
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Generation of Transgenic Mice
Six founders were initially identified from 50 live births resulting from pronuclear injection with the 2.9
tTA transgene. Two of these founders died suddenly between 3 and 5 weeks postnatally and were unavailable for further examination. A third founder failed to transmit the transgene. The three remaining lines were expanded by crossing with nontransgenic littermates.
Northern Blot Analysis
Expression of the tTA transgene was initially examined by Northern blot analysis of total cardiac RNA prepared from F1 offspring in 2.9tTA lines 6, 12, and 26. No expression was detected in line 26 (not shown). In line 6, several distinct bands and some diffuse hybridization were detected (Fig 2, left). The major hybridizing band was 1.5 kb, in agreement with the predicted size of the fully processed 2.9tTA transcript. The additional higher-molecular-weight hybridization signals may represent incomplete transcriptional termination within the transgene array. On longer exposure (Fig 2, right), a similar pattern of hybridization was also observed in line 12. The overall signal intensity in line 2.9tTA6 was substantially higher than in line 12, and therefore line 6 was primarily used for the subsequent studies.
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Baseline Transcription Is Variable
A total of 85 progeny resulting from matings between the 2.9tTA and LU5 transgenic strains were studied. Hearts were harvested either within 24 hours of birth (neonatal group) or 2 to 3 months after birth (adult group). A proportion of the neonatal mice were born to mothers that had been treated with tetracycline throughout gestation, and some of the adult mice were randomly selected to receive tetracycline-supplemented drinking water for at least 7 days prior to harvest. All progeny were genotyped for the presence or absence of the tTA and luciferase transgenes.
As shown in Fig 3 (top), only background levels of luciferase activity were detected in either group N or group T mice. Mice from group L, showed higher levels of reporter gene activity (P<.05, L versus T or N). Interestingly, the extent of luciferase activity in this group, which was tTA-independent, varied widely. In most mice, essentially no activity above background levels was detected, whereas in others, including siblings with the same transgenotype, substantially greater levels of reporter gene activity were observed.
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Transactivation and Repression
Introduction of the tTA transgene into the LU5 background markedly induced target gene expression (Fig 3, group D). Luciferase activity in the binary transgenics was almost 300-fold greater than in those mice harboring only the luciferase transgene (P<.05, D versus L). There was some variability in the responses of individual double-transgenic mice, which paralleled the heterogeneity observed in single-transgenic mice carrying only the luciferase target transgene.
Tetracycline treatment was highly effective in repressing the tTA-dependent transactivation. Compared with the untreated double-transgenic group, antibiotic therapy repressed the magnitude of luciferase activity in binary mice by >98% (D+ compared with D), to levels not significantly different from mice harboring only the LU5 transgene (P=.35, D+ compared with L). Thus, the proportion of luciferase activity that was dependent on the activity of the transactivator was completely repressed by tetracycline treatment.
The absolute magnitude of target gene expression in the hearts of binary-transgenic mice was determined by comparison to a standard curve generated with pure luciferase. On average, 5 ng of luciferase accumulated within each mouse heart, which corresponded to 3000 luciferase molecules per cardiocyte.
Stage and Tissue Specificity
One potentially important application of conditional gene expression is to achieve stage-specific expression for developmental studies. Therefore, we compared the behavior of the tetracycline-regulated system in newborns and adults. As shown in Fig 3 (bottom), the extent of background luciferase activity (group L), magnitude of transactivation (group D), and degree of repression (group D+) were not significantly different during different stages of development.
To determine the spatial specificity of conditional gene expression imparted by the MHC promoter, various tissues from mice carrying only the luciferase target gene or binary mice carrying both the target gene and tTA transgenes were assayed for luciferase activity. As previously reported, variable leakiness in the single-transgenic luciferase mice was seen in many tissues,13 but no statistically significant transactivation was observed in any of the tissues examined, including liver, kidney, spleen, tongue, or skeletal muscle (Fig 4). There was a trend toward significant transactivation in the lung (P=.12), which may reflect expression of the tTA transgene in the so-called "pulmonary myocardium," a location in which endogenous MHC expression in the mouse has been detected.34
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Target Gene Expression Within the Heart Is Heterogeneous
The spatial profile of tTA-dependent gene expression within the heart was examined using the G2 transgenic strain, which places a nuclear-localized lacZ target gene downstream of the chimeric tTA-responsive promoter. As expected, no ß-galactosidase activity was detected in the nuclei of nontransgenic mice or in those carrying just the 2.9tTA transgene (not shown). X-gal staining of the hearts of binary mice, carrying both the target and tTA transgenes, demonstrated abundant myocytes with deeply blue nuclei, indicative of transactivation of the lacZ target gene and expression of nuclear-localized ß-galactosidase activity (Fig 5A). In contrast to the more variable transcriptional leakiness observed in mice carrying the luciferase transgene, no tTA-independent expression was observed in any mice carrying only the lacZ target gene (Figs 5B and 5E). Treatment with doxycycline, a tetracycline analogue, for a period of 10 days completely abolished expression of the lacZ gene; virtually no blue nuclei were observed anywhere within the hearts of antibiotic-treated binary-transgenic mice (Figs 5C and 5F).
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Transactivation of the lacZ target gene in the hearts of doubly transgenic mice was heterogeneous. Expression was generally more uniform within the atria (Figs 5D and 5I) than within the ventricles (Figs 5G and 5H); however, in all chambers, cells with no evidence of ß-galactosidase activity were situated directly adjacent to others with intensely blue nuclei.
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Discussion |
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In summary, the results presented here characterize a cardiac-specific conditional gene expression system based on tetracycline-responsive promoters. Our studies allow us to comment on the following parameters of this system: the extent of transcriptional leakiness, the tissue specificity of transactivation, the magnitude and heterogeneity of transactivation, and the efficacy of tetracycline-dependent repression.
First, the extent of baseline transcription, or leakiness, using this system is highly variable. This variability is seen between strains and also within individual lines; therefore, it is likely to represent effects of both the integration site and modifier loci, respectively. For example, in a previous study with an Id1 target gene30 and in the current work examining the lacZ reporter gene, essentially no expression was seen in the absence of coexpressed transactivator. In contrast, our studies here and a previous report examining several individual lines of luciferase reporter gene mice31 demonstrate substantial differences in the magnitude of luciferase activity within single lines. To some extent, this apparent leakiness may represent the greater sensitivity of the luciferase assay than either Northern blot or histochemical analysis of lacZ expression. Nonetheless, the variability within individual lines is notable and is most likely due to differential inheritance and expression of modifier loci. Conceivably, experiments carried out using inbred genetic backgrounds, rather than the mixed genetic employed in these studies, might mitigate this variability, a possibility we are currently examining. Moreover, given the experience with both the Id1 and lacZ genes, as well as a recent report describing regulated expression of SV40 large T antigen,32 individual lines of target gene mice with functionally insignificant transcriptional leakiness can usually be identified.
Second, use of the MHC promoter to direct expression of the tetracycline-controlled transactivator results in essentially cardiac-specific target gene transactivation. This result is not surprising, based on the previous extensive characterization of various myosin heavy chain regulatory elements, both in vitro and particularly in previous transgenic experiments.16 In none of the tissues examined other than heart did we find significant transactivation. There was a trend to increased activity in the lung, which is explained by the normal presence of MHC in the pulmonary myocardium.34
On average, we found a 300-fold increase in luciferase activity in binary-transgenic mice harboring both the target gene and cardiac-specific transactivator compared with those carrying only the reporter gene. Because of the variable leakiness in some target gene mice, resulting in an average luciferase activity in single-transgenic mice that is greater than background, the 300-fold increase may represent an underestimation of the actual extent of transactivation that can be achieved. A more useful way of quantifying the magnitude of expression is on an absolute basis. By comparison to purified luciferase standards, we found that on average, about 5 ng of luciferase accumulated in the hearts of double-transgenic mice, equivalent to 3000 molecules per myocyte. By this measure, the accumulation of the target gene product is relatively modest, suggesting that regulatory proteins, particularly those with enzymatic activity, might be ideal. However, in view of the heterogeneous pattern of expression throughout the heart, accumulation within the subset of myocytes that successfully transactivate the target gene must be substantially greater than the average level. Moreover, the half-life of luciferase in vivo is relatively short,35 and more stable target proteins might accumulate to substantially greater levels. Nonetheless, our quantitative data suggest that regulatory proteins, particularly those with enzymatic activity, might be ideal targets for this system.
The nonuniform expression of the lacZ target gene is interesting, especially within the ventricles, where adjacent myocytes show divergent responses. This heterogeneity may reflect minor but functionally critical differences in the intranuclear concentration of tTA protein, which in some cells falls below the threshold necessary for efficient target gene transactivation. This possibility could best be addressed by immunohistochemical staining for tTA; unfortunately, no appropriately sensitive antibody reagents are currently available. Heterogeneous transgene expression in the heart has previously been reported with the 650-bp rat MHC promoter, where ectopic expression of skeletal troponin C was observed only in a subset of cardiac myocytes.36 Alternatively, this behavior may represent a stochastic, cell-autonomous inactivation of transgenic loci, an effect that is potentially exaggerated in a binary system. Although we have not examined the integration site of any of these transgenes, this behavior is reminiscent of position-effect variegation first described in Drosophila.37 Nonetheless, it is likely that expression of tTA using the 5-kb mouse MHC fragment described by Robbins' group might mitigate this heterogeneity.16
Certainly one critical parameter for a conditional genetic system is the capacity to virtually extinguish target gene expression. By this measure, the tetracycline-regulated system is quite effective. Both qualitatively, as visualized by the X-gal reaction, and quantitatively, by using the highly sensitive luciferase assay, target gene expression was reduced to levels indistinguishable from those observed in the absence of transactivator. While in some individual mice the level of expression in the presence of tetracycline was not insignificant, this behavior was no different from mice carrying only the target gene and does not reflect a failure to repress tTA-dependent transactivation.
In total, we have now generated ten lines of mice with tTA under the regulatory control of MHC sequences.30 None of those lines with the 0.6-kb promoter element gave persistent tTA expression or transactivation beyond the F2 generation. Among those lines generated with the 2.9-kb MHC promoter, we identified two that express the chimeric tTA protein at levels sufficient to transactivate effectively without transcriptional squelching and perinatal death. Thus, the identification of transgenic mice that express tTA within this functionally useful window is not trivial. Nonetheless, our goal of identifying and characterizing a stable line of cardiac-specific transactivators that might find widespread utility for conditional transgenic studies of the heart has been achieved.
In conclusion, these data demonstrate that a tetracycline-regulated gene expression system can be adapted to achieve highly regulated and tissue-specific expression of target genes in transgenic mice. While the quantitative and spatial data provide insights into potential limitations of this system in vivo, ongoing studies with tTA-dependent targets other than simple reporter genes suggest that novel phenotypes may successfully be generated with this system that are conditional in nature. Furthermore, the availability of one component of this binary regulatory system, ie, transgenic mice with stable, cardiac-specific expression of tTA, should be of substantial value for cardiovascular researchers, since only the target gene strain need be derived de novo. These and other advances in genetic manipulation,5 38 39 in combination with evolving methodologies to examine physiology in the intact mouse and isolated heart,13 15 40 41 42 should greatly enhance our understanding of cardiovascular development and function.
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Acknowledgments |
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This study was supported in part by grants from the American Heart Association, New York Affiliate and National Center. Dr Fishman is an Established Investigator of the American Heart Association. We wish to thank Bruce Conklin for helpful discussions and assistance, Bruce Markham for providing rat MHC
sequences, Lothar Hennighausen for the LU5 and G2 transgenic mice, and the Analytical Ultrastructure Center for technical assistance. Received June 7, 1996; accepted July 12, 1996.
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References |
|---|
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Camper SA, Saunders TL, Kendall SK, Keri RA, Seasholtz AF, Gordon DF, Birkmeier TS, Keegan CE, Karolyi IJ, Roller ML, et al. Implementing transgenic and embryonic stem cell technology to study gene expression, cell-cell interactions and gene function. Biol Reprod. 1995;52:246-257.[Abstract]
2. Iannaccone PM, Scarpelli DG. Exploring pathogenetic mechanisms using transgenic animals. Ann Med. 1993;25:131-138.[Medline] [Order article via Infotrieve]
3. Metsaranta M, Vuorio E. Transgenic mice as models for heritable diseases. Ann Med. 1992;24:117-120.[Medline] [Order article via Infotrieve]
4. Chien KR. Recent advances in the molecular biology of cardiac growth and hypertrophy: of mouse and man. Cardiologia. 1992;37:95-103.[Medline] [Order article via Infotrieve]
5.
Chien KR. Molecular advances in cardiovascular biology. Science. 1993;260:916-917.
6.
Chien KR. Cardiac muscle diseases in genetically engineered mice: evolution of molecular physiology. Am J Physiol. 1995;269:H755-H766.
7. Field LJ. Transgenic mice in cardiovascular research. Annu Rev Physiol. 1993;55:97-114.[Medline] [Order article via Infotrieve]
8. Lin MC, Rockman HA, Chien KR. Heart and lung disease in engineered mice. Nat Med. 1995;1:749-751.[Medline] [Order article via Infotrieve]
9. Paigen B, Plump AS, Rubin EM. The mouse as a model for human cardiovascular disease and hyperlipidemia. Curr Opin Lipidol. 1994;5:258-264.[Medline] [Order article via Infotrieve]
10.
Field LJ. Atrial natriuretic factor-SV40 T antigen transgenes produce tumors and cardiac arrhythmias in mice. Science. 1988;239:1029-1033.
11.
Hunter JJ, Tanaka N, Rockman HA, Ross J Jr, Chien KR. Ventricular expression of a MLC-2v-ras fusion gene induces cardiac hypertrophy and selective diastolic dysfunction in transgenic mice. J Biol Chem. 1995;270:23173-23178.
12.
Hunter JJ, Zhu H, Lee KJ, Kubalak S, Chien KR. Targeting gene expression to specific cardiovascular cell types in transgenic mice. Hypertension. 1993;22:608-617.
13.
Milano CA, Allen LF, Rockman HA, Dolber PC, McMinn TR, Chien KR, Bond RA, Lefkowitz RJ. Enhanced myocardial function in transgenic mice overexpressing the beta 2-adrenergic receptor. Science. 1994;264:582-586.
14. Rindt H, Subramaniam A, Robbins J. An in vivo analysis of transcriptional elements in the mouse alpha-myosin heavy chain gene promoter. Transgenic Res. 1995;4:397-405.[Medline] [Order article via Infotrieve]
15.
Rockman HA, Ono S, Ross RS, Jones LR, Karimi M, Bhargava V, Ross JJ, Chien KR. Molecular and physiological alterations in murine ventricular dysfunction. Proc Natl Acad Sci U S A. 1994;91:2694-2698.
16.
Subramaniam A, Jones WK, Gulick J, Wert S, Neumann J, Robbins J. Tissue-specific regulation of the alpha-myosin heavy chain gene promoter in transgenic mice. J Biol Chem. 1991;266:24613-24620.
17. Braun T, Rudnicki MA, Arnold HH, Jaenisch R. Targeted inactivation of the muscle regulatory gene Myf-5 results in abnormal rib development and perinatal death. Cell. 1992;71:369-382.[Medline] [Order article via Infotrieve]
18. Hasty P, Bradley A, Morris JH, Edmondson DG, Venuti JM, Olson EN. Muscle deficiency and neonatal death in mice with a targeted mutation in the myogenin gene. Nature. 1993;364:501-506.[Medline] [Order article via Infotrieve]
19. Rudnicki MA, Braun T, Hinuma S, Jaenisch R. Inactivation of MyoD in mice leads to up-regulation of the myogenic HLH gene Myf-5 and results in apparently normal muscle development. Cell. 1992;71:383-390.[Medline] [Order article via Infotrieve]
20. Rudnicki MA, Jaenisch R. The MyoD family of transcription factors and skeletal myogenesis. Bioessays. 1995;17:203-209.[Medline] [Order article via Infotrieve]
21. Rudnicki MA, Schnegelsberg PN, Stead RH, Braun T, Arnold HH, Jaenisch R. MyoD or Myf-5 is required for the formation of skeletal muscle. Cell. 1993;75:1351-1359.[Medline] [Order article via Infotrieve]
22.
Venuti JM, Morris JH, Vivian JL, Olson EN, Klein WH. Myogenin is required for late but not early aspects of myogenesis during mouse development. J Cell Biol. 1995;128:563-576.
23.
Hanks M, Wurst W, Anson-Cartwright L, Auerbach AB, Joyner AL. Rescue of the En-1 mutant phenotype by replacement of En-1 with En-2. Science. 1995;269:679-682.
24. Wang Y, Schnegelsberg P, Dausman J, Jaenisch R. Functional redundancy of the muscle-specific transcription factors Myf5 and myogenin. Nature. 1996;379:823-825.[Medline] [Order article via Infotrieve]
25.
Baim S, Labow M, Levine A, Shenk T. A chimeric mammalian transactivator based on the lac repressor that is regulated by temperature and isopropyl-D-thiogalactopyranoside. Proc Natl Acad Sci U S A. 1991;88:5072-5076.
26. Fishman G. Conditional transgenics. Trends Cardiovasc Med. 1996;5:211-217.
27.
Ornitz D, Moreadith R, Leder P. Binary system for regulating transgene expression in mice: targeting int-2 gene expression with yeast GAL4/UAS control elements. Proc Natl Acad Sci U S A. 1991;88:698-702.
28. Ringold G. Regulation of mouse mammary tumor virus gene expression by glucocorticoid hormones. Curr Top Microbiol Immunol. 1996;106:79-103.
29.
Gossen M, Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci U S A. 1992;89:5547-5551.
30. Passman RS, Fishman GI. Regulated expression of foreign genes in vivo after germline transfer. J Clin Invest. 1994;94:2421-2425.
31.
Furth PA, St Onge L, Boger H, Gruss P, Gossen M, Kistner A, Bujard H, Hennighausen L. Temporal control of gene expression in transgenic mice by a tetracycline-responsive promoter. Proc Natl Acad Sci U S A. 1994;91:9302-9306.
32.
Efrat S, Fusco-DeMane D, Lemberg H, al Emran O, Wang X. Conditional transformation of a pancreatic beta-cell line derived from transgenic mice expressing a tetracycline-regulated oncogene. Proc Natl Acad Sci U S A. 1995;92:3576-3580.
33. Fishman GI, Kaplan ML, Buttrick PM. Tetracycline-regulated cardiac gene expression in vivo. J Clin Invest. 1994;93:1864-1868.
34. Jones WK, Sanchez A, Robbins J. Murine pulmonary myocardium: developmental analysis of cardiac gene expression. Dev Dyn. 1994;200:117-128.[Medline] [Order article via Infotrieve]
35. Thompson J, Hayes L, Lloyd D. Modulation of firefly luciferase stability and impact on studies of gene regulation. Gene. 1991;103:171-177.[Medline] [Order article via Infotrieve]
36.
McDonald KS, Field LJ, Parmacek MS, Soonpaa M, Leiden JM, Moss RL. Length dependence of Ca2+ sensitivity of tension in mouse cardiac myocytes expressing skeletal troponin C. J Physiol. 1995;483:131-139.
37. Reuter G, Spierer P. Position effect variegation and chromatin proteins. Bioessays. 1992;14:605-612.[Medline] [Order article via Infotrieve]
38. Fuller SJ, Chien KR. Genetic engineering of cardiac muscle cells: in vitro and in vivo. Genet Eng. 1994;16:17-31.
39. Robbins J. Gene targeting: the precise manipulation of the mammalian genome. Circ Res. 1993;73:3-9.[Abstract]
40.
Grupp IL, Subramaniam A, Hewett TE, Robbins J, Grupp G. Comparison of normal, hypodynamic, and hyperdynamic mouse hearts using isolated work-performing heart preparations. Am J Physiol. 1993;265:H1401-H1410.
41.
Hewett TE, Grupp IL, Grupp G, Robbins J. -Skeletal actin is associated with increased contractility in the mouse heart. Circ Res. 1994;74:740-746.
42.
Ng WA, Grupp IL, Subramaniam A, Robbins J. Cardiac myosin heavy chain mRNA expression and myocardial function in the mouse heart. Circ Res. 1991;68:1742-1750.
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 B. Gellen, M. Fernandez-Velasco, F. Briec, L. Vinet, K. LeQuang, P. Rouet-Benzineb, J.-P. Benitah, M. Pezet, G. Palais, N. Pellegrin, et al. Conditional FKBP12.6 Overexpression in Mouse Cardiac Myocytes Prevents Triggered Ventricular Tachycardia Through Specific Alterations in Excitation- Contraction Coupling Circulation, April 8, 2008; 117(14): 1778 - 1786. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. May, D. Gilon, V. Djonov, A. Itin, A. Lazarus, O. Gordon, C. Rosenberger, and E. Keshet Transgenic system for conditional induction and rescue of chronic myocardial hibernation provides insights into genomic programs of hibernation PNAS, January 8, 2008; 105(1): 282 - 287. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. T. McCloskey, S. Turcato, G.-Y. Wang, L. Turnbull, B.-Q. Zhu, T. Bambino, A. P. Nguyen, D. H. Lovett, R. A. Nissenson, J. S. Karliner, et al. Expression of a Gi-coupled receptor in the heart causes impaired Ca2+ handling, myofilament injury, and dilated cardiomyopathy Am J Physiol Heart Circ Physiol, January 1, 2008; 294(1): H205 - H212. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Sainte-Marie, A. N. D. Cat, R. Perrier, L. Mangin, C. Soukaseum, M. Peuchmaur, F. Tronche, N. Farman, B. Escoubet, J.-P. Benitah, et al. Conditional glucocorticoid receptor expression in the heart induces atrio-ventricular block FASEB J, October 1, 2007; 21(12): 3133 - 3141. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Kehat, R. Heinrich, O. Ben-Izhak, H. Miyazaki, J. S. Gutkind, and A. Aronheim Inhibition of basic leucine zipper transcription is a major mediator of atrial dilatation Cardiovasc Res, June 1, 2006; 70(3): 543 - 554. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. G. Trivieri, G. Y. Oudit, R. Sah, B.-G. Kerfant, H. Sun, A. O. Gramolini, Y. Pan, A. D. Wickenden, W. Croteau, G. Morreale de Escobar, et al. Cardiac-specific elevations in thyroid hormone enhance contractility and prevent pressure overload-induced cardiac dysfunction PNAS, April 11, 2006; 103(15): 6043 - 6048. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Turnbull, H.-Z. Zhou, P. M. Swigart, S. Turcato, J. S. Karliner, B. R. Conklin, P. C. Simpson, and A. J. Baker Sustained preconditioning induced by cardiac transgenesis with the tetracycline transactivator Am J Physiol Heart Circ Physiol, March 1, 2006; 290(3): H1103 - H1109. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Lee, R. Agah, M. Xiao, A. D. Frutkin, M. Kremen, H. Shi, and D. A. Dichek Regulated Transgene Expression in Vascular Smooth Muscle Circ. Res., October 14, 2005; 97(8): e85 - e85. [Full Text] [PDF]
|
|
|
|
|
|
S. Handa, M. A. Momen, A.-M. Sadi, T. Afroze, C. Wang, and M. Husain Troubles With a Transgene: Experiences With SM22{alpha}-tTA Mice Circ. Res., October 14, 2005; 97(8): e85 - e87. [Full Text] [PDF]
|
|
|
|
|
|
L. Barandon, P. Dufourcq, P. Costet, C. Moreau, C. Allieres, D. Daret, P. D. Santos, J.-M. D. Lamaziere, T. Couffinhal, and C. Duplaa Involvement of FrzA/sFRP-1 and the Wnt/Frizzled Pathway in Ischemic Preconditioning Circ. Res., June 24, 2005; 96(12): 1299 - 1306. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. T. McCloskey, L. Turnbull, P. M. Swigart, A. C. Zambon, S. Turcato, S. Joho, W. Grossman, B. R. Conklin, P. C. Simpson, and A. J. Baker Cardiac transgenesis with the tetracycline transactivator changes myocardial function and gene expression Physiol Genomics, June 16, 2005; 22(1): 118 - 126. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Ouvrard-Pascaud, Y. Sainte-Marie, J.-P. Benitah, R. Perrier, C. Soukaseum, A. N. D. Cat, A. Royer, K. Le Quang, F. Charpentier, S. Demolombe, et al. Conditional Mineralocorticoid Receptor Expression in the Heart Leads to Life-Threatening Arrhythmias Circulation, June 14, 2005; 111(23): 3025 - 3033. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Skurk, Y. Izumiya, H. Maatz, P. Razeghi, I. Shiojima, M. Sandri, K. Sato, L. Zeng, S. Schiekofer, D. Pimentel, et al. The FOXO3a Transcription Factor Regulates Cardiac Myocyte Size Downstream of AKT Signaling J. Biol. Chem., May 27, 2005; 280(21): 20814 - 20823. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. L. Yang, R. Gros, M. G. Kabir, A. Sadi, A. I. Gotlieb, M. Husain, and D. J. Stewart Conditional Cardiac Overexpression of Endothelin-1 Induces Inflammation and Dilated Cardiomyopathy in Mice Circulation, January 20, 2004; 109(2): 255 - 261. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Barandon, T. Couffinhal, P. Dufourcq, J. Ezan, P. Costet, D. Daret, C. Deville, and C. Duplaa Frizzled A, a novel angiogenic factor: promises for cardiac repair Eur. J. Cardiothorac. Surg., January 1, 2004; 25(1): 76 - 83. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. P. Czubryt and E. N. Olson Balancing Contractility and Energy Production: The Role of Myocyte Enhancer Factor 2 (MEF2) in Cardiac Hypertrophy Recent Prog. Horm. Res., January 1, 2004; 59(1): 105 - 124. [Abstract] [Full Text]
|
|
|
|
|
|
S. Kovacic, C.-L. M. Soltys, A. J. Barr, I. Shiojima, K. Walsh, and J. R. B. Dyck Akt Activity Negatively Regulates Phosphorylation of AMP-activated Protein Kinase in the Heart J. Biol. Chem., October 10, 2003; 278(41): 39422 - 39427. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. N. Mungrue, D. J. Stewart, and M. Husain The Janus Faces of iNOS Circ. Res., October 3, 2003; 93 (7): e74 - e74. [Full Text] [PDF]
|
|
|
|
 |
|
 W. Schubert, X. Y. Yang, T. T.C. Yang, S. M. Factor, M. P. Lisanti, J. D. Molkentin, M. Rincon, and C.-W. Chow Requirement of transcription factor NFAT in developing atrial myocardium J. Cell Biol., June 9, 2003; 161(5): 861 - 874. [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]
|
|
|
|
|
|
M. P. Czubryt, J. McAnally, G. I. Fishman, and E. N. Olson Regulation of peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha ) and mitochondrial function by MEF2 and HDAC5 PNAS, February 18, 2003; 100(4): 1711 - 1716. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Most, A. Remppis, and H. A Katus Conditional AC type VI expression in the heart: relevant insights into function of inducible target gene expression Cardiovasc Res, November 1, 2002; 56(2): 181 - 183. [Full Text] [PDF]
|
|
|
|
|
|
M. H. Gao, H. Bayat, D. M Roth, J. Yao Zhou, J. Drumm, J. Burhan, and H Kirk Hammond Controlled expression of cardiac-directed adenylylcyclase type VI provides increased contractile function Cardiovasc Res, November 1, 2002; 56(2): 197 - 204. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. T. Beggah, B. Escoubet, S. Puttini, S. Cailmail, V. Delage, A. Ouvrard-Pascaud, B. Bocchi, M. Peuchmaur, C. Delcayre, N. Farman, et al. From the Cover: Reversible cardiac fibrosis and heart failure induced by conditional expression of an antisense mRNA of the mineralocorticoid receptor in cardiomyocytes PNAS, May 14, 2002; 99(10): 7160 - 7165. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Bjornsson, E. Andersson, P. Lundstrom, N. Larsson, X. Xu, E. Repetowska, R. K. Humphries, and S. Karlsson Proliferation of primitive myeloid progenitors can be reversibly induced by HOXA10 Blood, December 1, 2001; 98(12): 3301 - 3308. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Nerbonne, C. G. Nichols, T. L. Schwarz, and D. Escande Genetic Manipulation of Cardiac K+ Channel Function in Mice: What Have We Learned, and Where Do We Go From Here? Circ. Res., November 23, 2001; 89(11): 944 - 956. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Suzuki, W.-J. Shen, B. D. Nelson, S. Patel, J. H. Veerkamp, S. P. Selwood, G. M. Murphy Jr., E. Reaven, and F. B. Kraemer Absence of cardiac lipid accumulation in transgenic mice with heart-specific HSL overexpression Am J Physiol Endocrinol Metab, October 1, 2001; 281(4): E857 - E866. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. J. Baker, C. H. Redfern, M. D. Harwood, P. C. Simpson, and B. R. Conklin Abnormal contraction caused by expression of Gi-coupled receptor in transgenic model of dilated cardiomyopathy Am J Physiol Heart Circ Physiol, April 1, 2001; 280(4): H1653 - H1659. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y Dor, T. Camenisch, A Itin, G. Fishman, J. McDonald, P Carmeliet, and E Keshet A novel role for VEGF in endocardial cushion formation and its potential contribution to congenital heart defects Development, January 5, 2001; 128(9): 1531 - 1538. [Abstract] [PDF]
|
|
|
|
 |
|
 F. JAISSER Inducible Gene Expression and Gene Modification in Transgenic Mice J. Am. Soc. Nephrol., November 1, 2000; 11(90002): S95 - S100. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. H. Redfern, M. Y. Degtyarev, A. T. Kwa, N. Salomonis, N. Cotte, T. Nanevicz, N. Fidelman, K. Desai, K. Vranizan, E. K. Lee, et al. Conditional expression of a Gi-coupled receptor causes ventricular conduction delay and a lethal cardiomyopathy PNAS, April 25, 2000; 97(9): 4826 - 4831. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. S. Williams and P. D. Wagner Transgenic animals in integrative biology: approaches and interpretations of outcome J Appl Physiol, March 1, 2000; 88(3): 1119 - 1126. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. C. Tardiff, T. E. Hewett, S. M. Factor, K. L. Vikstrom, J. Robbins, and L. A. Leinwand Expression of the beta (slow)-isoform of MHC in the adult mouse heart causes dominant-negative functional effects Am J Physiol Heart Circ Physiol, February 1, 2000; 278(2): H412 - H419. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Lee, G. Morley, Q. Huang, A. Fischer, S. Seiler, J. W. Horner, S. Factor, D. Vaidya, J. Jalife, and G. I. Fishman Conditional lineage ablation to model human diseases PNAS, September 15, 1998; 95(19): 11371 - 11376. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. I. Fishman Timing Is Everything in Life : Conditional Transgene Expression in the Cardiovascular System Circ. Res., May 4, 1998; 82(8): 837 - 844. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. F. James, T. E. Hewett, and J. Robbins Cardiac Physiology in Transgenic Mice Circ. Res., March 9, 1998; 82(4): 407 - 415. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. James and J. Robbins Molecular remodeling of cardiac contractile function Am J Physiol Heart Circ Physiol, November 1, 1997; 273(5): H2105 - H2118. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. J. Alden, P. H. Goldspink, S. W. Ruch, P. M. Buttrick, and J. Garcia Enhancement of L-type Ca2+ current from neonatal mouse ventricular myocytes by constitutively active PKC-beta II Am J Physiol Cell Physiol, April 1, 2002; 282(4): C768 - C774. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. S. Sohal, M. Nghiem, M. A. Crackower, S. A. Witt, T. R. Kimball, K. M. Tymitz, J. M. Penninger, and J. D. Molkentin Temporally Regulated and Tissue-Specific Gene Manipulations in the Adult and Embryonic Heart Using a Tamoxifen-Inducible Cre Protein Circ. Res., July 6, 2001; 89(1): 20 - 25. [Abstract] [Full Text] [PDF]
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