TECHNOLOGY REPORT

Inducible Cardiomyocyte-Specific Gene Disruption Directedby the Rat Tnnt2 Promoter in the MouseBingruo Wu,1 Bin Zhou,2,3 Yidong Wang,1 Hsiu-Ling Cheng,4 Calvin T. Hang,4 William T. Pu,2,3

Ching-Pin Chang,4 and Bin Zhou1,5,6*1Department of Genetics, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York2Department of Cardiology, Children’s Hospital Boston, Boston, Massachusetts3Department of Genetics, Harvard Medical School, Boston, Massachusetts4Division of Cardiovascular Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, California5Department of Medicine, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York6Department of Pediatrics, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York

Received 22 July 2009; Revised 3 September 2009; Accepted 9 September 2009

Summary: We developed a conditional and induciblegene knockout methodology that allows effective genedeletion in mouse cardiomyocytes. This transgenicmouse line was generated by coinjection of two trans-genes, a ‘‘reverse’’ tetracycline-controlled transactivator(rtTA) directed by a rat cardiac troponin T (Tnnt2) pro-moter and a Cre recombinase driven by a tetracycline-responsive promoter (TetO). Here, Tnnt2-rtTA activatedTetO-Cre expression takes place in cardiomyocytesfollowing doxycycline treatment. Using two differentmouse Cre reporter lines, we demonstrated that expres-sion of Cre recombinase was specifically and robustlyinduced in the cardiomyocytes of embryonic or adulthearts following doxycycline induction, thus, allowingcardiomyocyte-specific gene disruption and lineagetracing. We also showed that rtTA expression and doxy-cycline treatment did not compromise cardiac function.These features make the Tnnt2-rtTA;TetO-Cre trans-genic line a valuable genetic tool for analysis of spatio-temporal gene function and cardiomyocyte lineagetracing during developmental and postnatal periods.genesis 48:63–72, 2010. VVC 2009 Wiley-Liss, Inc.

Key words: cardiomyocyte; Cre recombinase; doxycycline;rtTA; Tnnt2

INTRODUCTION

Cardiac-specific gene disruption in the mouse hasadvanced our current understanding of the biologicalfunction of gene products in mammalian heart develop-ment (Robbins, 2004). Mouse cardiac gene recombina-tion involves the use of a cardiomyocyte-specific Credeletor and a floxed allele of a gene of interest. The effi-cacy and precision of cardiac recombination, therefore,depends on the expression level and tissue specificity ofCre recombinase. Since the first cardiac-specific Credeletor line was generated (Agah et al., 1997) using apromoter of the mouse cardiac myosin heavy chain

(MHC) (Gulick et al., 1991), new Cre deletors have beencreated to improve recombination specificity andefficiency. For example, to avoid potential transgenicinsertion artifacts and to achieve ventricular myocar-dium-specific gene inactivation, a knock-in strategy wasused in which Cre recombinase was inserted into themouse myosin light chain 2v (MLC2v) genomic locus(Chen et al., 1998). In addition to cardiac structuralgenes, the genomic locus or the promoter/enhancer ofNkx2.5, which encodes a transcription factor essentialfor cardiogenesis (Lyons et al., 1995), was used to gener-ate Cre lines for analysis of gene function in Nkx2.5-expressing cardiac cell lineages (McFadden et al., 2005;Moses et al., 2001; Stanley et al., 2002).

The major advantage of tissue-specific recombinationusing Cre recombinase is to avoid embryonic lethalitycaused by germline loss-of-function mutations that pre-cludes analysis of gene function in postnatal hearts.However, spatial control of gene deletion alone may notbe sufficient for studying cardiac function in later embry-onic development or in the postnatal period when car-diac-specific gene deletion still results in severe cardiacdefects and early embryonic lethality. Techniques thatenable temporal control of gene deletion in the develop-ing and postnatal hearts have been developed using atetracycline-controlled transactivator (tTA) driven by arat MHC promoter (Fishman et al., 1994; Yu et al.,

* Correspondence to: Bin Zhou, MD, PhD, Department of Genetics,

Albert Einstein College of Medicine of Yeshiva University, Price Center 420,

1301 Morris Park Avenue, Bronx, NY 10461.

E-mail: bin.zhou@einstein.yu.edu

Contract grant sponsors: American Heart Association, NIH, Tobacco-

Related Disease Research Program, Turner-Hazinski Award from Vanderbilt

Children’s Hospital, Vanderbilt UniversityPublished online 15 December 2009 in

Wiley InterScience (www.interscience.wiley.com).

DOI: 10.1002/dvg.20573

' 2009 Wiley-Liss, Inc. genesis 48:63–72 (2010)

1996), or by an attenuated mouse MHC promoter (Sanbeet al., 2003).

In the tTA or ‘‘Tet-off’’ system, transgene expression isrepressed by a continuous long-term doxycycline (Dox)treatment. Removal of Dox activates transgene expres-sion or initiates Cre-mediated recombination (Zhu et al.,2002). However, chronic Dox treatment may have sideeffects on cardiac function and gene expression. Doxmay accelerate the onset of cardiac hypertrophy and theprogression to congestive heart failure in mice (Vinetet al., 2008). Furthermore, Dox has a long half-life inmice (Robertson et al., 2002), therefore, removal of Doxdoes not allow prompt induction of gene deletion. Suchdisadvantages can be minimized by using a ‘‘Tet-on’’ sys-tem or a ‘‘reverse’’ tTA (rtTA) converted from the tTA bypoint mutations (Freundlieb et al., 1999), in which trans-gene expression is active in the presence of Dox. A dou-bly transgenic system that drives expression of rtTAfrom tissue-specific promoters and activates Cre recom-binase expression directed by a doxycycline/rtTA-induci-

ble promoter has emerged as a leading approach permit-ting tight spatiotemporal control of gene deletion inmany tissues (Branda and Dymecki, 2004).

A single transgenic mouse harboring both rtTA andCre transgenes will provide a useful genetic tool for tem-poral analysis of gene function in cardiomyocytes andtheir lineage evolution. Therefore, we generated a newinducible cardiomyocyte-specific Cre mouse line usingrtTA under the control of a rat cardiac troponin T(Tnnt2) promoter (Wang et al., 2000). The choice ofthis promoter over the mouse MHC promoter was basedon previous observations that Tnnt2-Cre resulted in anearlier and more uniform recombination in the develop-ing heart (Chen et al., 2006; Jiao et al., 2003) than thatinduced by MHC-Cre (Agah et al., 1997).

We made the Tnnt2-rtTA driver construct in thepWhere plasmid (InvivoGen, San Diego, CA), which con-tains two mouse H19 insulators (mH19) protecting theflanked Tnnt2-rtTA cassette from potential positioneffects on transgene expression (Fig. 1a). The responsive

FIG. 1. Generation of Tnnt2-rtTA;TetO-Cre doubly transgenic mice. (a) Schematic of Tnnt2-rtTA and TetO-Cre transgenic constructs. (b)Schematic of inducible Cre expression directed by Dox and the doubly transgenic system. (c) Example of PCR genotyping shows co-segre-gation of rtTA and Cre transgenes in the established transgenic line. (d) Example of RT-PCR reveals cardiac-specific expression of rtTAwhereas the cardiac-specific expression of Cre recombinase is Dox-dependent. H, K, Li, Lm, Lu; heart, kidney, liver, limb, lung. [Color figurecan be viewed in the online issue,which is available at www.interscience.wiley.com.]

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nuclear-localized Cre (nls-Cre) construct was generatedusing the TetO-CMV promoter (Pan et al., 2000). Thetwo genetic constructs were simultaneously microin-jected into fertilized eggs of FVB/N mice to generatefounders that were then tested for Cre expression uponDox treatment (Fig. 1b). Eight doubly transgenic found-ers were obtained from the microinjection. Seven ofeight founders had both constructs and the transgenestatus of the offspring was analyzed by PCR (Fig. 1c). Wefound that the two transgenes consistently cosegregatedand passed on to the offspring in a Mendelian fashion inall seven lines, suggesting that the constructs insertedinto the host genome at the same location. We showedby RT-PCR analysis that rtTA expression was restrictedto the embryonic heart in two lines. Cre expression wasonly detected in the embryonic heart after Dox induc-tion, indicating that Cre expression was tightly con-trolled by the ‘‘Dox-rtTA’’ and not leaky (Fig. 1d). Thus,rtTA expression is restricted to the heart, and its induc-tion of cardiac-specific Cre activity requires Doxtreatment.

We tested the feasibility of these two lines for induc-ing genomic recombination by crossing them with theR26-floxstop-lacZ reporter line (R26R) (Soriano, 1999).One line (t26) gave reliable recombination after Doxtreatment. This line was backcrossed with C57BL/6 forfive generations and maintained in this background forrest of the studies. We found that 1-day induction withDox in the drinking water (1 mg ml21) at either em-bryonic Day (E) 9, 10, 11, or 11.5 was sufficient toCre-mediated excision of the floxed stop codon andactivation of lacZ reporter expression in the wholeheart at E10, 11, 11.5, or 12.5 (Fig. 2a–e). Gene recom-bination was not observed outside of the heart afteran overnight X-gal staining, demonstrating a cardiac-specific induction of Cre expression in these embryos.Significant recombination was also found at later em-bryonic stages with 1-day Dox treatment, although therecombination was not as efficient. In contrast, a 2-daytreatment greatly improved induction efficiency andled to complete gene recombination in older embryos(Fig. 2f,g). Cardiac gene recombination was not foundin embryos without Dox induction, indicating no orundetectable ‘‘leakiness’’ of Cre expression (Fig. 2h).Analysis of histological sections confirmed that cardiac-specific induction of Cre-mediated recombination wasrobust throughout the myocardium of the entire heartand restricted to cardiomyocytes (Fig. 2i–I). No re-porter expression was found in the mesenchymal cellsof the outflow tract (oft), atrioventricular cannel (avc)(Fig. 2i,j), or the epicardium (Fig. 2k). Additional analy-sis showed complete gene recombination in 1-week-old neonatal hearts when Dox was given to nursingfemales for 3 days starting at the day of delivery (datanot shown). Taken together, these observations dem-onstrate that the Tnnt2-rtTA;TetO-Cre transgenic meth-odology allows efficient induction of Cre-mediatedgene recombination in the cardiomyocytes of bothembryonic and neonatal hearts.

To assess whether this transgenic method is also appli-cable for inducible gene recombination in adult hearts,we performed a serial of analyses of Tnnt2-rtTA;TetO-Creadult mice using a double-fluorescent Cre reporter line(RosaR/G) that contains a membrane-targeted tandemdimmer Tomato (mT), a red fluorescent protein (RFP),and a floxstop-membrane-targeted green fluorescentprotein (mG) (Muzumdar et al., 2007) (Fig. 3a). TheRosa

R/G line also uses a strong enhancer, whichimproves expression of reporter genes in the postnatalheart (Muzumdar et al., 2007). We found that Dox induc-tion (1 mg ml21 in the drinking water) for 5 daysresulted in significant activation of GFP expression inthe hearts of triple compound heterozygous mice(Tnnt2rtTA/1;TetOCre/1

;RosaR/G/1) (Fig. 3b). Attenuation

of RFP intensity of the whole heart also indicated inacti-vation of its expression in cardiomyocytes but not innoncardiomyocytes such as the endothelial, epithelial,smooth muscle, or fibroblast cells. In contrast, controllittermates without the Tnnt2-rtTA;TetO-Cre transgenesexpressed only RFP, but not GFP (Fig. 3c). In the absenceof Dox, there was no detectable GFP expression ineither triple compound heterozygous (Fig. 3d) or controlnontransgenic mice (Fig. 3e), indicating that the recom-bination was Dox-dependent.

We next used coimmunohistochemistry to analyze thecell-specificity of gene recombination directed by theTnnt2-rtTA in adult hearts in the presence of Dox. Byusing an antibody against GFP and antibodies that markspecifically endothelial or smooth muscle cells, weshowed that gene recombination was clearly cardiomyo-cyte-specific, as Cre-mediated GFP expression did notoverlap with expression of Pecam1 (Fig. 4a, arrow) orFlk1 (Fig. 4b, arrow) in endothelial cells. Furthermore,the recombination was not observed in smooth musclecells, which were labeled by SM-actin (Fig. 4c, arrow-head) or SM22 antibodies (Fig. 4d, arrowhead).

We also employed double immunostaining with anti-bodies against GFP or several markers of cardiomyocytesto study if they were marked by Tnnt2-rtTA;TetO-Cre-mediated gene recombination. The staining showedthat recombination was indeed restricted to cardiomyo-cytes, as expression of the GFP reporter colocalizedwith desmin (Fig. 5a,b), Tnnt2 (Fig. 5c), and a2-actinin(Actn2) (Fig. 5d). Overall, these findings demonstratethat expression of the Tnnt2-rtTA transgene is restrictedto cardiomyocytes, and that in the presence of Dox, itcan effectively activate the TetO promoter to direct Cre--mediated recombination specifically in cardiomyocytes.

To assess the effect of transgenes on cardiac function,we analyzed the viability and cardiac physiology ofTnnt2-rtTA;TetO-Cre mice. These mice treated with Doxand with lifetime cardiac rtTA expression appearedgrossly normal, healthy, and fertile. Their lifespan wascomparable to their nontransgene littermates up to atleast 9 months of age when the breeders were retiredfrom experimental use (Fig. 6a). To determine cardiacfunction in the Tnnt2-rtTA;TetO-Cre mice, we performedtreadmill exercise tests and echocardiographic analysis.

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FIG. 2. Tnnt2-rtTA directs efficient recombination during heart development in the R26-floxstop-lacZ reporter line (R26R). (a–g) A panel ofphotos of wholemount X-gal staining shows the cardiac-specific Cre-mediated recombination of the floxstop lacZ and beta-galactosidaseexpression in embryonic hearts after Dox induction. Note that no sign of recombination is outside of the heart. (h) A photo of wholemountX-gal staining shows no beta-galactosidase expression in the doubly transgenic heart without Dox induction. (i–l) Panel of histological sec-tions shows cardiac-specific recombination (beta-galactosidase expression) in embryonic hearts. Note that no beta-galactosidase expres-sion in the nonmyocardial mesenchymal outflow tract (oft) and atrioventricular cannel (avc) at E10.5 and E12.5 as well as in the epicardiumat E14.5 (arrows).

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We showed that transgenic mice and control littermatesat the age of 6–8 months with short-term (1 week) Doxtreatment had comparable exercise duration, indicatingthat rtTA expression does not limit cardiac function andendurance (Fig. 6b). Furthermore, echocardiographicanalysis revealed that transgenic and nontransgenic miceexhibited similar values of the left ventricular internaldiameter both in diastole (LVIDd) and in systole (LVIDs)(Fig. 6c). The left ventricular ejection fraction (LVEF)and left ventricular fractional shortening (LVFS) were

also comparable (Fig. 6d). These cardiac functionalmeasurements indicate that rtTA expression and Doxtreatment have no appreciable effects on cardiacfunction.

In summary, we have generated and characterizeda new Tnnt2-rtTA;TetO-Cre transgenic mouse line thatallows temporally controlled and tissue-specific generecombination in the embryonic, perinatal, and adulthearts. At desirable time points, short-term Doxtreatment efficiently activates gene recombination in

FIG. 3. Tnnt2-rtTA drives effective recombination and activation of the double-fluorescent Cre reporter (RosaR/G) in the adult heart. (a)Schematic shows that Tnnt2-rtTA induces cardiac-specific recombination and expression of a membrane-targeted GFP (mG) in the RosaR/G

reporter line also constitutively expresses membrane-associated Tomato RFP (mT). (b) Fluorescent photos show significant GFP expressionin hearts of the triple compound heterozygous mice (Tnnt2rtTA/1;TetOCre/1;RosaR/G/1) after a 5-day Dox induction. Continuous but attenu-ated RFP expression in the entire heart was from non-cardiomyocytes after Dox treatment. (c) No GFP expression was found in nontrans-genic control mice [Tg(-)] with the same treatment. (d) Tnnt2-rtTA without Dox induction was unable to activate GFP expression. (e) Photosshow no GFP expression in control mice [Tg(-)] without Dox treatment. Bar 5 200 um. The far right panels show the sectional view of theareas (indicated by the white box) on the left. Bar5 50 lm.

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cardiomyocytes for investigating gene functions in theheart. This transgenic method is also useful for label-ing cardiomyocyte lineages. These features make thistransgenic line a useful genetic tool for spatiotempo-ral studies of gene functions in cardiomyocytes dur-ing embryonic heart development, perinatal heartmaturation, and postnatal cardiac physiology andpathology.

MATERIALS AND METHODS

Animal Maintenance and Treatment

Maintenance of mice and animal experiments were

performed according to protocols approved by the

Institutional Animal Care and Use Committee of

Albert Einstein College of Medicine, Vanderbilt Uni-

FIG. 4. Tnnt2-rtTA directed recombination is specific for cardiomyocytes. (a,b) Immunohistochemistry shows that the Tnnt2-rtTA-directed,Cre-mediated GFP expression (grey) is not in the endothelial cells, which express cell surface markers Pecam1 and Flk1 (red, indicated byarrow). (c,d) The Tnnt2-rtTA directed Cre-mediated GFP expression (grey or green) is not in smooth muscle cells, which express smoothmuscle cell actin (SM-Actin) and SM22 (red, indicated by arrowhead). Bar5 50 lm.

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versity, Boston Children’s Hospital, and Stanford Uni-versity. R26R and Rosa

R/G/1 mice were purchasedfrom Jackson Laboratory (Bar Harbor, MN). Noontimeon the day of detecting vaginal plugs was designatedas E0.5. Dox (Sigma-Aldrich, St. Louis, MO) treatmentwas carried out in the drinking water at a concentra-tion of 1 mg ml21 and provided to the pregnantfemale mice at different gestation time points, nurs-ing females, or adult mice. For adult mice, Dox

water was administrated for 5–7 days, and changedevery other day.

Generation of the Tnnt2-rtTA;TetO-Cre TransgenicLine

The Tnnt2-rtTA driver construct was made by insert-ing a 0.5-kb Tnnt2 promoter into the pWhere in vivoreporter plasmid between Sda I and Nco I sites, and a1.0-kb nuclear localized (nls) rtTA cDNA into Nco I and

FIG. 5. Tnnt2-rtTA-directed recombination marks cardiomyocyte lineages. (a,b) Immunohistochemistry shows that the Tnnt2-rtTA-directed, Cre-mediated GFP expression (red in a or green in b) in Desmin-expressing cardiomyocytes [grey in (a) or red in (b)]. (c) Tnnt2-rtTAdirected Cre-mediated GFP expression (green) in Tnnt2-expressing cardiomyocytes (red). (d) Tnnt2-rtTA directed Cre-mediated GFPexpression (green) in the a2-actinin (Actn2)-expressing cardiomyocytes (red). Bar5 50 lm.

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Nhe I sites (Fig. 1a). The pWhere plasmid containstwo H19 insulators, which we have previouslyshown are able to effectively prevent ectopic tran-scription influences from random insertion sites(Zhou et al., 2005). The responsive Cre constructwas generated by cloning an nls-Cre into a TetO-CMV

promoter plasmid between BamH I sites in PBS (Panet al., 2000). The constructs were confirmed by en-zymatic digestion and sequencing. Transgenic mice

were generated by coinjection of the two constructsinto fertilized eggs from FVB/N mice.

Transgenic founder mice were genotyped by PCRanalysis on tail tip genomic DNA using primers for rtTAand Cre. The PCR-positive founder mice were crossedwith C57BL/6 mice, and transgenic offsprings wereidentified by PCR. Primer pairs used for genotypingincluded 50-rtTA: 50-TCGACGCCTTAGCCATTGAGAT-30,30-rtTA: 50GGCTGTACGCGGACCCACTTTC-30, yielding

FIG. 6. Cardiac expression of rtTA and Dox treatment has no adverse effect on cardiac function. (a) Schematic shows comparable viabilitybetween transgenic mice (Tg) and wild-type (WT) littermates within the first 9 months. (b) A graph shows comparable exercise endurancebetween transgenic mice and wild-type littermates in treadmill tests. (c) Graphs of echocardiographic measurements show normal cardiacfunction in transgenic mice and wild-type littermates. LVIDd, left ventricular internal diameter in diastole; LVIDs, left ventricular internaldiameter in systole; LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening. [Color figure can be viewed in theonline issue,which is available at www.interscience.wiley.com.]

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476-bp PCR product; and 50-Cre: 50-GGCGCGGCAACACCATTTTT-30, 30-Cre: 50-TCCGGGCTGCCACGACCAA-30,producing 446-bp PCR product.

X-Gal Staining

Wholemount X-gal staining was carried out asdescribed before (Zhou et al., 2005). Embryos from E9.5to E12.5, or the hearts isolated at later stages, were fixedin 4% paraformaldehyde/PBS on ice for 30 min to 2 haccording to the stages, washed in PBS, and then stainedwith fresh prepared X-gal solution for 2 h at 378C or over-night at 308C. After staining, the samples were washedwith PBS, post-fixed, and processed for histological analy-sis or cleared in a glycerol gradient before being photo-graphed under a stereoscope (SMZ800, Nikon, Melville,NY).

Immunofluorescence and Immunohistochemistry

Mouse hearts (3 to 4-month old) were excised in halfand fixed in 4% PFA for 4 h at 48C, followed by a PBSwash at 48C. Hearts were soaked in a sucrose gradient(10–30%) in PBS before embedding in OCT. Sections (8lm) were obtained using a cryostat, collected on poly-L-lysine-coated slides and fixed in 1:1 methanol/acetonefor 10 min at 2208C. Staining was performed accordingto protocols as described (Zhou et al., 2006). Primaryantibodies used in these experiments were GFP(A21311, Invitrogen, Carlsbad, CA), Desmin (213M, Bio-meda, Foster City, CA), Actn2 (A7811, Sigma, St. Louis,MO), Tnnt2 (MS-295, Lab Vision, Fremont, CA), Pecam 1(553370, BD Pharmingen, San Jose, CA), Flk1 (NB600-1433, Novus, Littleton, CO), SM-Actin (C6198, Sigma,St. Louis, MO), and SM-22 (Ab14106, Abcam, Cambridge,MA). Secondary antibodies used were conjugated withAlexa Fluor fluorescent dyes (Invitrogen, Carlsbad, CA)or HRP and developed with a chromagenic systemaccording to the manufacturer’s protocol (Vector Lab,Burlingame, CA). The immunostained tissues wereimaged using confocal microscopy (FV1000, Olympus,Center Valley, PA).

Analysis of Cardiac Function

Treadmill exercise was carried out as follows. Trans-genic mice at 6–8 months of age were placed into tread-mill chambers 10 min before starting to run to allowacclimation. Starting speed was 7.5 m min21 and startingincline was 48. Every 3 min, speed was increased by 2.5m min21, and incline was increased by 28. When speedreached 20 m min21, only the incline was increased.The workout was terminated when mice were laggingand stayed on the electric grid for 10 s. Cardiac functionand structure of 6- to 8-month old transgenic and controlwild-type mice was also examined by echocardiogramafter 1-week of Dox treatment using the Vevo 770machine (VisualSonics Toronto, Ontario, Canada). Statis-tic analysis was performed using the Student t-test(unpaired). The value P < 0.05 was designated as havingsignificant difference between the testing groups.

ACKNOWLEDGMENTS

The authors thank Kai Jiao and Chi-Wing Chow for pro-viding plasmids for these studies; Kevin Tompkins fortechnical support in pronuclear injection; and BerniceMorrow and Laina Freyer for comments on the manu-script. This work was initiated at Vanderbilt University,continued and finished at Albert Einstein College ofMedicine.

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