Cardiac-specific gene disruption in the mouse has advanced our current understanding of the biological function of gene products in mammalian heart development (Robbins, 2004
). Mouse cardiac gene recombination involves the use of a cardiomyocyte-specific Cre deletor and a floxed allele of a gene of interest. The efficacy and precision of cardiac recombination, therefore, depends on the expression level and tissue specificity of Cre recombinase. Since the first cardiac-specific Cre deletor line was generated (Agah et al., 1997
) using a promoter of the mouse cardiac myosin heavy chain (MHC) (Gulick et al., 1991
), new Cre deletors have been created to improve recombination specificity and efficiency. For example, to avoid potential transgenic insertion artifacts and to achieve ventricular myocardium-specific gene inactivation, a knock-in strategy was used in which Cre recombinase was inserted into the mouse myosin light chain 2v (MLC2v
) genomic locus (Chen et al., 1998
). In addition to cardiac structural genes, the genomic locus or the promoter/enhancer of Nkx2.5
, which encodes a transcription factor essential for cardiogenesis (Lyons et al., 1995
), was used to generate Cre lines for analysis of gene function in Nkx2.5-expressing cardiac cell lineages (Moses et al., 2001
) (Stanley et al., 2002
) (McFadden et al., 2005
The major advantage of tissue-specific recombination using Cre recombinase is to avoid embryonic lethality caused by germline loss-of-function mutations that precludes analysis of gene function in postnatal hearts. However, spatial control of gene deletion alone may not be sufficient for studying cardiac function in later embryonic development or in the postnatal period when cardiac-specific gene deletion still results in severe cardiac defects and early embryonic lethality. Techniques that enable temporal control of gene deletion in the developing and postnatal hearts have been developed using a tetracycline-controlled transactivator (tTA) driven by a rat MHC
promoter (Fishman et al., 1994
; Yu et al., 1996
), or by an attenuated mouse MHC
promoter (Sanbe et al., 2003
In the tTA or “Tet-off” system, transgene expression is repressed by a continuous long-term doxycycline (Dox) treatment. Removal of Dox activates transgene expression or initiates Cre-mediated recombination (Zhu et al., 2002
). However, chronic Dox treatment may have side effects on cardiac function and gene expression. Dox may accelerate the onset of cardiac hypertrophy and the progression to congestive heart failure in mice (Vinet et al., 2008
). Furthermore, Dox has a long half-life in mice (Robertson et al., 2002
), therefore, removal of Dox does not allow prompt induction of gene deletion. Such disadvantages can be minimized by using a “Tet-on” system or a “reverse” tTA (rtTA) converted from the tTA by point mutations (Freundlieb et al., 1999
), in which transgene expression is active in the presence of Dox. A doubly transgenic system that drives expression of rtTA from tissue-specific promoters and activates Cre recombinase expression directed by a doxycycline/rtTA-inducible promoter has emerged as a leading approach permitting tight spatiotemporal control of gene deletion in many tissues (Branda and Dymecki, 2004
A single transgenic mouse harboring both rtTA
transgenes will provide a useful genetic tool for temporal analysis of gene function in cardiomyocytes and their lineage evolution. Therefore, we generated a new inducible cardiomyocyte-specific Cre mouse line using rtTA under the control of a rat cardiac troponin T (Tnnt2
) promoter (Wang et al., 2000
). The choice of this promoter over the mouse MHC
promoter was based on previous observations that Tnnt2
-Cre resulted in an earlier and more uniform recombination in the developing heart (Jiao et al., 2003
) (Chen et al., 2006
) than that induced by MHC
-Cre (Agah et al., 1997
We made the Tnnt2-rtTA
driver construct in the pWhere plasmid (InvivoGen, San Diego, CA), which contains two mouse H19
insulators (mH19) protecting the flanked Tnnt2-rtTA
cassette from potential position effects on transgene expression (). The responsive nuclear-localized Cre (nls-Cre) construct was generated using the TetO-CMV
promoter (Pan et al., 2000
). The two genetic constructs were simultaneously microinjected into fertilized eggs of FVB/N mice to generate founders that were then tested for Cre expression upon Dox treatment (). Eight doubly transgenic founders were obtained from the microinjection. Seven of eight founders had both constructs and the transgene status of the offspring was analyzed by PCR (). We found that the two transgenes consistently co-segregated and passed on to the offspring in a Mendelian fashion in all 7 lines, suggesting that the constructs inserted into the host genome at the same location. We showed by RT-PCR analysis that rtTA expression was restricted to the embryonic heart in two lines. Cre expression was only detected in the embryonic heart after Dox induction, indicating that Cre expression was tightly controlled by the `Dox-rtTA' and not leaky (). Thus, rtTA expression is restricted to the heart, and its induction of cardiac-specific Cre activity requires Dox treatment.
Generation of Tnnt2-rtTA;TetO-Cre doubly transgenic mice
We tested the feasibility of these two lines for inducing genomic recombination by crossing them with the R26-floxstop-lacZ
reporter line (R26R) (Soriano, 1999
). One line (t26) gave reliable recombination after Dox treatment. This line was backcrossed with C57BL/6 for 5 generations and maintained in this background for rest of the studies. We found that one-day induction with Dox in the drinking water (1 mg/ml) at either embryonic day (E) 9, 10, 11, or 11.5 was sufficient to Cre-mediated excision of the floxed stop codon and activation of lacZ
reporter expression in the whole heart at E10, 11, 11.5, or 12.5 (). Gene recombination was not observed outside of the heart after an overnight X-gal staining, demonstrating a cardiac-specific induction of Cre expression in these embryos. Significant recombination was also found at later embryonic stages with one-day Dox treatment, although the recombination was not as efficient. In contrast, a 2-day treatment greatly improved induction efficiency and led to complete gene recombination in older embryos (). Cardiac gene recombination was not found in embryos without Dox induction, indicating no or undetectable “leakiness” of Cre expression (). Analysis of histological sections confirmed that cardiac-specific induction of Cre-mediated recombination was robust throughout the myocardium of the entire heart and restricted to cardiomyocytes (). No reporter expression was found in the mesenchymal cells of the outflow tract (oft), atrioventricular cannel (avc) (), or the epicardium (). Additional analysis showed complete gene recombination in one-week old neonatal hearts when Dox was given to nursing females for 3 days starting at the day of delivery (data not shown). Taken together, these observations demonstrate that the Tnnt2-rtTA;TetO-Cre
transgenic methodology allows efficient induction of Cre-mediated gene recombination in the cardiomyocytes of both embryonic and neonatal hearts.
Tnnt2-rtTA directs efficient recombination during heart development in the R26-floxstop-lacZ reporter line (R26R)
To assess whether this transgenic method is also applicable for inducible gene recombination in adult hearts, we performed a serial of analyses of Tnnt2-rtTA;TetO-Cre
adult mice using a double-fluorescent Cre reporter line (RosaR/G
) that contains a membrane-targeted tandem dimmer Tomato (mT), a red fluorescent protein (RFP), and a floxstop-membrane-targeted green fluorescent protein (mG) (Muzumdar et al., 2007
) (). The RosaR/G
line also uses a strong enhancer, which improves expression of reporter genes in the postnatal heart (Muzumdar et al., 2007
). We found that Dox induction (1 mg/ml in the drinking water) for 5 days resulted in significant activation of GFP expression in the hearts of triple compound heterozygous mice (Tnnt2rtTA/+;TetOCre/+;RosaR/G/+
) (). Attenuation of RFP intensity of the whole heart also indicated inactivation of its expression in cardiomyocytes but not in non-cardiomyocytes such as the endothelial, epithelial, smooth muscle, or fibroblast cells. In contrast, control littermates without theTnnt2-rtTA;TetO-Cre
transgenes expressed only RFP, but not GFP (). In the absence of Dox, there was no detectable GFP expression in either triple compound heterozygous () or control non-transgenic mice (), indicating that the recombination was Dox-dependent.
Tnnt2-rtTA drives effective recombination and activation of the double-fluorescent Cre reporter (RosaR/G) in the adult heart
We next used co-immunohistochemistry to analyze the cell-specificity of gene recombination directed by the Tnnt2-rtTA in adult hearts in the presence of Dox. By using an antibody against GFP and antibodies that mark specifically endothelial or smooth muscle cells, we showed that gene recombination was clearly cardiomyocyte-specific, as Cre-mediated GFP expression did not overlap with expression of Pecam1 (, arrow) or Flk1 (, arrow) in endothelial cells. Furthermore, the recombination was not observed in smooth muscle cells, which were labeled by SM-actin (, arrowhead) or SM22 antibodies (, arrowhead).
Tnnt2-rtTA directed recombination is specific for cardiomyocytes
We also employed double immunostaining with antibodies against GFP or several markers of cardiomyocytes to study if they were marked by Tnnt2-rtTA;TetO-Cre-mediated gene recombination. The staining showed that recombination was indeed restricted to cardiomyocytes, as expression of the GFP reporter co-localized with desmin (), Tnnt2 (), and α2-actinin (Actn2) (). Overall, these findings demonstrate that expression of the Tnnt2-rtTA transgene is restricted to cardiomyocytes, and that in the presence of Dox, it can effectively activate the TetO promoter to direct Cre-mediated recombination specifically in cardiomyocytes.
Tnnt2-rtTA-directed recombination marks cardiomyocyte lineages
To assess the effect of transgenes on cardiac function, we analyzed the viability and cardiac physiology of Tnnt2-rtTA;TetO-Cre mice. These mice treated with Dox and with lifetime cardiac rtTA expression appeared grossly normal, healthy and fertile. Their lifespan was comparable to their non-transgene littermates up to at least 9 months of age when the breeders were retired from experimental use (). To determine cardiac function in the Tnnt2-rtTA;TetO-Cre mice, we performed treadmill exercise tests and echocardiographic analysis. We showed that transgenic mice and control littermates at the age of 6 to 8 months with short-term (1 week) Dox treatment had comparable exercise duration, indicating that rtTA expression does not limit cardiac function and endurance (). Furthermore, echocardiographic analysis revealed that transgenic and non-transgenic mice exhibited similar values of the left ventricular internal diameter both in diastole (LVIDd) and in systole (LVIDs) (). The left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were also comparable (). These cardiac functional measurements indicate that rtTA expression and Dox treatment have no appreciable effects on cardiac function.
Cardiac expression of rtTA and Dox treatment has no adverse effect on cardiac function
In summary, we have generated and characterized a new Tnnt2-rtTA;TetO-Cre transgenic mouse line that allows temporally controlled and tissue-specific gene recombination in the embryonic, perinatal, and adult hearts. At desirable time points, short-term Dox treatment efficiently activates gene recombination in cardiomyocytes for investigating gene functions in the heart. This transgenic method is also useful for labeling cardiomyocyte lineages. These features make this transgenic line a useful genetic tool for spatiotemporal studies of gene functions in cardiomyocytes during embryonic heart development, perinatal heart maturation, and post-natal cardiac physiology and pathology.