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(Circulation Research. 2001;88:587.)
© 2001 American Heart Association, Inc.

Integrative Physiology

Inducible Gene Targeting in Postnatal Myocardium by Cardiac-Specific Expression of a Hormone-Activated Cre Fusion Protein

Tetsuo Minamino, Vinciane Gaussin, Francesco J. DeMayo, Michael D. Schneider

From the Center for Cardiovascular Development (T.M., V.G., M.D.S.) and Departments of Medicine (T.M., V.G., F.J.D., M.D.S.), Molecular and Cellular Biology (M.D.S.), and Molecular Physiology and Biophysics (M.D.S.), Baylor College of Medicine, Houston, Tex. Present address of V.G. is Cardiovascular Research Institute, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, and Hackensack University Medical Center, Hackensack, NJ.

Correspondence to Dr Michael D. Schneider, Center for Cardiovascular Development, Baylor College of Medicine, One Baylor Plaza, Room 506C, Houston, TX 77030. E-mail michaels{at}bcm.tmc.edu

	Abstract

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Abstract—Cardiac-restricted expression of Cre recombinase can provoke lineage-specific gene excision in the myocardium. However, confounding early lethality may still preclude using loss-of-function models to study the postnatal heart. Here, we have tested whether inducible, heart-specific recombination can be triggered after birth by transgenic expression of a Cre fusion protein that incorporates a mutated progesterone receptor ligand binding domain (PR1) that is activated by the synthetic antiprogestin, RU486, but not by endogenous steroid hormones. CrePR1 driven by the -myosin heavy chain (MHC) promoter was expressed specifically in heart. Translocation of CrePR1 from cytoplasm to nuclei in ventricular myocytes was induced by RU486. To establish whether this approach can mediate cardiac-specific, drug-dependent excision between loxP sites in vivo, we mated MHC-CrePR1 mice with a ubiquitously expressed (ROSA26) Cre reporter line. Offspring harboring MHC-CrePR1 and/or the floxed allele were injected with RU486 versus vehicle, and the prevalence of ß-galactosidase (ß-gal)–positive cells was determined, indicative of Cre-mediated excision. Little or no baseline recombination was seen 1 week after birth. Cardiac-restricted, RU486-inducible recombination was demonstrated in bigenic mice at age 3 and 6 weeks, using each of 3 independent CrePR1 lines. Recombination in the absence of ligand paralleled the levels of CrePR1 protein expression and was more evident at 6 weeks. Thus, conditional, posttranslational activation of a Cre fusion protein can bypass potential embryonic and perinatal effects on the heart and permits inducible recombination in cardiac muscle. High levels of the chimeric Cre protein, in particular, were associated with progressive recombination in the absence of drug.

Key Words: Cre recombinase • genetics • progesterone receptor • transgenic mice

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Gene targeting to generate loss-of-function mutations in the mouse has yielded remarkable advances in understanding the roles played by specific gene products.¹ Although insights have been gained into both embryogenesis and later gene function, inherent impediments including indirect systemic defects can complicate the use of homologous recombination in the germline to elicit the role of genes in a specific pathophysiological context. In the face of these limitations, the possibility of somatic, tissue-specific recombination has gained appeal. One strategy for this exploits the Cre/loxP system derived from bacteriophage P1.^{2 3} The 38-kDa Cre protein functions as a site-specific recombinase, splicing DNA between specific 34-bp sequences known as loxP sites. Mating mice that contain a loxP-flanked gene with mice expressing Cre under the control of a heart-specific promoter results in Cre-mediated excision selectively in cardiac muscle.^{4 5 6}

However, confounding premature lethality may still exist, if gene recombination were to have severe effects at earlier stages than desired, and long-term, chronic compensation can also occur. Because the Cre/loxP system is entirely contingent on the exogenous recombinase, recombination can be regulated via the timing and tissue specificity of Cre transgene expression.^{7 8} We previously showed, by adenoviral gene transfer to adult myocardium, that even postmitotic cardiac muscle cells are amenable to Cre-mediated recombination.⁴ An alternative strategy has been attempted for cell type–specific plus temporal control, by tissue-restricted expression of a conditionally functional chimeric Cre protein, comprising a fusion between Cre and the mutated ligand binding domain (LBD) of a steroid hormone receptor.^{7 8 9 10 11} These chimeric proteins become selectively active on binding a synthetic ligand, eg, RU486 or tamoxifen in preference to endogenous progesterone and estrogen, respectively. An attractive extension of this technology would confine Cre-mediated recombination to the postnatal myocardium. Toward this end, we have tested whether ligand-inducible DNA recombination can be achieved in the postnatal myocardium by posttranslational activation of Cre, using a progesterone receptor fusion protein expressed under the control of the cardiac-specific -myosin heavy chain (MHC) promoter.

	Materials and Methods

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Transgenic Mice
The MHC-CrePR1 vector was constructed using a 1.8-kb fragment of pCrePR1,⁷ kindly provided by G. Schutz (German Cancer Research Center, Heidelberg, Germany), which contains nuclear localization signal Cre, the mutated LBD of human progesterone receptor (amino acids 641 to 891),¹² and the simian virus (SV) 40 polyadenylation signal. CrePR1 was subcloned 3' to the 5.5-kb mouse MHC gene promoter,¹³ provided by J. Robbins (Children’s Hospital Research Foundation, Cincinnati, Ohio). The linearized MHC-CrePR1 gene was microinjected into the pronuclei of FVB/N zygotes at a concentration of 2 ng/µL, and injected embryos were transferred to pseudopregnant ICR females. To confirm transgene integration, genomic DNA was extracted from mice tails, digested with KpnI, and subjected to Southern blot analysis. Copy number was quantified by comparison with the recombinant plasmid as standard.

Cell Culture and Transfection
Ventricular myocytes from 2-day-old Sprague-Dawley rats (Charles River Laboratory) were isolated, purified by Percoll density gradient centrifugation, and preplated for 1 hour to enrich further for cardiac myocytes (>95%).¹⁴ Cells were plated at a density of 9x10⁵ cells per 35-mm well for gene transfer. Transfection was carried out using lipofectamine, 1.0 µg of the loxP-tagged CAG-CATZ reporter,¹⁵ 0.5 µg of an SV40-driven luciferase reporter gene to correct for transfection efficiency, and 2.0 µg of the test vector (the MHC promoter alone, MHC promoter-driven Cre [MHC-Cre],⁴ or MHC-CrePR1). Cells then were cultured in serum-free medium as described,¹⁴ with the addition of 1 nmol/L triiodothyronine to augment MHC transcription. Twenty-four hours after transfection, RU486 or 80% ethanol, the vehicle control, was added (2 µL, for 2 mL of medium), and cells were harvested 36 hours later.

Luciferase activity was monitored as the oxidation of luciferin in the presence of coenzyme A, using an Analytical Luminescence model 2010 luminometer.¹⁶ LacZ activity was determined using chlorophenol red–D-galactosidase as substrate.¹⁷ Total protein was measured by the Bradford method.¹⁸

Western Blot Analysis
For immunodetection of CrePR1, hearts and other organs from MHC-CrePR1 transgenic mice were frozen in liquid nitrogen, homogenized, and lysed in 20 mmol/L Tris-HCl, pH 8.0, containing 0.1% SDS, 0.5% NP-40, 1 mmol/L EDTA, 0.5% sodium deoxycholate, 40 µg/mL PMSF, 50 µmol/L leupeptin, and 50 µmol/L aprotinin. Cell lysates (50 µg of protein per lane) were resolved by 10% SDS-PAGE and transferred onto a nitrocellulose membrane (Schleicher & Schuell). The membrane was blocked in 5% nonfat milk in 0.05% Tween-20/Tris-buffered saline (TBS) for 1 hour at room temperature. After washing, the membrane was then incubated with polyclonal rabbit antibody against Cre¹⁹ (BAbCO; 1:500 in 1% nonfat milk in Tween-20/TBS) at 4°C overnight. After washing, the membrane was incubated with horseradish peroxidase–conjugated donkey antibody against rabbit IgG (Amersham Pharmacia Biotech Inc; 1:2000). Protein expression was visualized with enhanced chemiluminescence reagents (Amersham Pharmacia Biotech Inc).

Polymerase Chain Reaction (PCR) Analysis
MHC CrePR1 transgenic mice were identified by amplification of genomic DNA from tail samples using the forward primer 5'-ATGACAGACAGATCCCTCCTATCTCC-3' and reverse primer 5'-CTCATCACTCGTTGCATCATCGAC-3'. ROSA26 Cre reporter mice,²⁰ provided by P. Soriano (Fred Hutchinson Cancer Research Center, Seattle, Wash), were identified using the forward primer 5'-CGCCATCCCGCATCTGACCAC-3' and reverse primer 5'-CCGCTCTGCTACCTGCGCC-3'. To monitor Cre-mediated recombination by PCR after transient cotransfection, DNA was extracted from cultured cardiac myocytes as described, using primers AG and Z3 for recombination of the pCAG-CATZ reporter.⁴ For PCR analysis of Cre-mediated recombination of the ROSA26 Cre reporter gene in vivo, the primers R26FA²⁰ and Z3 were used. As a positive control, ß-casein was amplified as described.⁴ All PCR reactions were as follows: 1 cycle at 94°C for 2 minutes, followed by 32 cycles each at 94°C for 1 minute, 60°C for 1 minute, and 72°C for 1 minute, and 1 additional cycle at 72°C for 10 minutes.

Histochemistry
For histological detection and intracellular localization of Cre recombinase,^{11 21} hearts were fixed overnight in 4% paraformaldehyde. Sections were blocked overnight with TBS containing 2% BSA and 0.05% Triton X-100, washed 3 times, and then incubated overnight with the anti-Cre antibody (BAbCO, 1:100) in blocking solution at 4°C in a humidifying chamber. Bound primary antibody was visualized using FITC-conjugated antibody against rabbit IgG. Nuclei were stained with 2 µg/mL DAPI, and cardiac myocytes were identified using MF20 antibody to sarcomeric MHC (1 µg/mL; University of Iowa Hybridoma Bank) followed by Texas Red–conjugated antibody against mouse IgG (Molecular Probes).¹⁴

To quantify Cre-dependent recombination using ß-gal expression, hearts were harvested from 3- or 6-week old animals after intraperitoneal administration of RU486 versus the vehicle for 5 days (0.25 and 0.60 mg/d, respectively, or sesame oil alone). Hearts were fixed for 1.5 hours at 4°C in 2% formaldehyde, 0.2% glutaraldehyde, 0.2% NP-40, 5 mmol/L ethylenebis(oxyethylenenitrilo)tetraacetic acid, 2 mmol/L MgCl₂, and 0.1 mol/L sodium phosphate (pH 7.4); incubated in 0.5 mol/L sucrose; mounted in freezing medium; and frozen in 100% ethanol on dry ice. Sets of 6- to 8-µm cryostat sections were obtained and used for 5-bromo-4-chloro-3-indolyl-D-galactopyranoside (X-Gal) staining. To estimate the percentage of X-Gal–positive cells, >600 cells were scored per condition, using 5 fields from 3 serial sections of each of 2 to 3 animals.⁴ Images were captured with a Zeiss Axioplan 2 epifluorescence microscope. Cre-dependent ß-gal expression also was visualized by whole-mount X-Gal staining of isolated organs as described.⁴

Statistics
Data are expressed as mean±SE. Results were compared by ANOVA followed by the Student-Newman-Keuls multiple-comparison test, using a significance level of P<0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
The Cre–Progesterone Receptor Fusion Protein Mediates RU486-Dependent Recombination in Cultured Ventricular Muscle Cells
To achieve cardiac myocyte–specific expression of Cre recombinase cDNA in frame with a mutated LBD of the human progesterone receptor, CrePR1⁷ was placed under the transcriptional control of the cardiac-specific MHC promoter¹³ (Figure 1A). CrePR1 is activated preferentially by the synthetic antiprogestin RU486, as opposed to native steroid hormones.^{7 8 9} As initial validation studies, cultured ventricular myocytes were cotransfected with the Cre-dependent reporter gene CAG-CATZ, together with vector containing the MHC promoter alone, MHC-Cre, or MHC-CrePR1, in the presence or absence of RU486. CAG-CATZ harbors a chloramphenicol acetyltransferase (CAT) gene flanked by loxP sites and driven by the chicken ß-actin promoter¹⁵ ; downstream of CAT is the Escherichia coli ß-gal gene (lacZ; Figure 1B). In the absence of Cre recombinase activity, the CAT gene prevents readthrough expression of lacZ; conversely, when the CAT gene is excised by Cre-mediated recombination between the tandem loxP sites, lacZ becomes positioned adjacent to the ß-actin promoter, permitting lacZ expression. Transient transfection of MHC-Cre provoked gene recombination in ventricular myocytes, equally, regardless of the presence or absence of RU486 (Figure 1C). This indicates that RU486 does not fortuitously influence expression or function of the conventional cardiac-specific Cre gene. By contrast, MHC-CrePR1 induced minimal lacZ activity in the absence of RU486, similar to that evoked by the MHC promoter alone, whereas the addition of RU486 increased lacZ activity to 60% of that elicited by MHC-Cre (Figure 1C). Thus, recombination mediated by the Cre fusion protein is tightly regulated by RU486 in ventricular myocytes in short-term culture.

View larger version (25K): [in this window] [in a new window]   Figure 1. RU486-inducible recombination in cultured cardiac myocytes, using a Cre–progesterone receptor fusion protein. A, Schematic representation of MHC-CrePR1 vector. B, Schematic representation of pCAG-CATZ. PCR primers used to monitor recombination, AG and Z3, flank the paired loxP sites and internal CAT gene. C, Analysis of Cre-mediated recombination by lacZ expression. Neonatal rat ventricular myocytes were cotransfected with 1 µg of pCAG-CATZ and 0.5 µg of an SV40-driven luciferase reporter gene (to correct for transfection efficiency), in the presence or absence of a Cre expression vector. Results (mean±SE; n=6 for each condition) are adjusted for the cotransfected luciferase gene and expressed relative to those elicited by plasmid containing the MHC promoter alone. D, Analysis of Cre-mediated recombination by PCR. Cardiac myocytes were transfected with pCAG-CATZ, MHC-Cre, and MHC-CrePR1 as indicated and were assayed for the appearance of the recombination-dependent 690-bp fragment.

To substantiate the above results by a more sensitive method, Cre-mediated recombination between the loxP sites was also assessed by PCR (Figure 1D). No recombination was detected when cardiac myocytes were transfected with CAG-CATZ, MHC-Cre, or MHC-CrePR1 alone. A recombination-dependent 690-bp fragment could be detected when cardiac myocytes were transfected with CAG-CATZ in combination with MHC-Cre. Within the limits of detection for 35 cycles of PCR, recombination of the CAG-CATZ reporter in the presence of MHC-CrePR1 was strictly contingent on the presence of RU486.

RU486 Induces Nuclear Translocation of the Cre–Progesterone Receptor Fusion Protein in Mouse Myocardium
To establish the feasibility of cardiac-specific inducible recombination in vivo, the MHC-CrePR1 construct was used to generate transgenic mice (Figure 2). Transgene-positive mice were identified by PCR. Four of 21 potential F₀ founders carried the MHC-CrePR1 gene, of whom 3 transmitted the gene. Southern blotting, by comparison with known amounts of the CrePR1 vector, estimated copy number to be 1 to 10 in the 3 established lines (Figure 2A). Cre protein expression was confirmed in myocardium of each line by Western blot analysis and was roughly proportional with copy number (Figure 2C); specificity of the rabbit anti-Cre antibody was demonstrated using 293 cells transiently transfected with a cytomegalovirus (CMV)–Cre or control expression vector (Figure 2B). By Western blot analysis, transgene expression was exclusive to the heart and was not detected in the other organs assayed (Figure 2D).

View larger version (40K): [in this window] [in a new window]   Figure 2. Construction of MHC-CrePR1 transgenic mice. A, Dot-blot analysis of copy number in 3 independent lines. B, Western blot analysis of transiently transfected 293 cells, indicating specificity of the anti-Cre antibody. - indicates control vector; +, CMV-Cre, indicating the expected 38-kDa protein. C, Western blot analysis of myocardium from the 3 transgenic lines and a transgene-negative control, showing relative levels of CrePR1. D, Western blot analysis, showing cardiac-restricted expression of the 57-kDa CrePR1 fusion protein (line 6279).

To characterize the translocation of CrePR1 after drug administration, immunohistochemical analysis was performed on ventricular sections from MHC-CrePR1 mice at the age of 3 weeks (Figure 3). Antibodies to Cre and DAPI were used to visualize the CrePR1 fusion protein and cell nuclei, respectively, and antibody to sarcomeric MHC was used to identify cardiac muscle cells. In the absence of ligand, CrePR1 protein was seen diffusely throughout the cytoplasm of ventricular myocytes. By contrast, RU486 induced the nuclear localization of CrePR1.

View larger version (66K): [in this window] [in a new window]   Figure 3. RU486-induced translocation of CrePR1 from cytoplasm to nucleus in ventricular myocardium. RU486 was administered by intraperitoneal injection (0.25 mg/d for 5 days, for the 3-week-old mice represented here). Immunohistochemistry was performed using antibodies to Cre and to sarcomeric MHC, followed by fluorochrome-conjugated antibodies to rabbit (green) and mouse (red) IgG, respectively; DAPI was used to visualize nuclei (blue).

RU486 Induces Cardiac-Specific Recombination in MHC-CrePR1xROSA26 Cre Reporter Mice
To evaluate whether RU486 can trigger conditional recombination via the CrePR1 fusion protein in the myocardium in vivo, MHC-CreP1 mice were mated with ROSA26 Cre reporter mice. In this reporter line, lacZ is expressed only after Cre-mediated excision of a loxP-flanked neomycin cassette.²⁰ To demonstrate the presence and tissue specificity of Cre-dependent recombination, MHC-CrePR1 mice, ROSA26 Cre reporter mice, and bigenic mice inheriting both genes were analyzed by whole-organ staining and PCR (Figure 4). No lacZ activity was seen in myocardium from mice inheriting either gene alone, or at 1 week in the myocardium of MHC-CrePR1xRosa26 Cre mice; RU486 was not administered at this age, to avoid manipulation of mice before weaning. In bigenic mice at the age of 3 weeks, whole-mount X-gal staining for 3 hours showed the marked induction of lacZ by treatment with RU486, with little spontaneous recombination. Although basal recombination was more readily detected at 6 weeks, administration of RU486 markedly increased lacZ staining at this age as well. Sporadic tubular lacZ staining was discernible in the lung, consistent with the known activity of the MHC promoter in pulmonary veins,¹³ and was increased by RU486. Results illustrated in Figure 4A are for line 6297.

View larger version (34K): [in this window] [in a new window]   Figure 4. RU486-inducible recombination in postnatal myocardium. A, Whole-organ X-Gal staining for lacZ expression, using the transgenic lines and ages indicated. Bar=5 mm. B, Tissue specificity of RU486-induced recombination, analyzed by PCR. Mice bearing MHC-CrePR1, the ROSA26 reporter gene, or both were analyzed after administration of RU486. Top, Structure of loxP-flanked gene segment in ROSA26 Cre reporter mice. PGK neo indicates phosphoglycerate kinase promoter-driven neomycin phosphotransferase gene. Bottom, PCR analysis of DNA from representative mice at the age of 6 weeks, indicating amplification of the recombination-dependent fragment. ß-Casein was used as a positive control. H indicates heart; Lu, lung; Li, liver; S, spleen; and Q, skeletal muscle (quadriceps).

Cre-mediated recombination was also analyzed by PCR in RU486-treated mice inheriting MHC-CrePR1, the ROSA26 Cre reporter gene, or both (Figure 4B). Recombination was confirmed only in bigenic mice, and was cardiac-specific, within the limits of sensitivity for the assay. The results illustrated are for line 6306, with similar findings for all 3 lines.

Quantitative assessment of the prevalence for recombination was performed by X-gal staining of ventricular sections from all 3 lines (Figure 5). Line 6306, with the lowest level of expression by Western blot (Figure 2), likewise showed the lowest prevalence of ligand-independent recombination, which increased from 11±1% to 58.5±3.2% with RU486. In this one line, the prevalence of X-gal staining in the atria was markedly diminished compared with the ventricles (not shown), which may reflect insertional rather than dosage effects, given the known activity of the MHC promoter in atrial myocardium.¹³ Marked induction of recombination by RU486 was also shown in 6-week-old mice harboring both genes, although spontaneous recombination in the absence of RU486 was increased in a time-dependent manner.

View larger version (70K): [in this window] [in a new window]   Figure 5. Prevalence of RU486-inducible recombination in postnatal myocardium shown by X-Gal staining for lacZ expression, using transgenic lines and ages indicated. Counterstaining was performed with Nuclear Fast Red. Bar=40 µm. Left, Representative data. Right, Mean results.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The capacity to trigger site-specific gene recombination selectively in cardiac muscle cells using the Cre/loxP system, or in other defined components of the cardiovascular system, has obvious attractiveness as a means to circumvent irrelevant lethality due to extraneous organs, preclude cardiovascular phenotypes that are merely the consequence of global or systemic defects, and pinpoint intrinsic ("cell-autonomous") versus nonautonomous mechanisms for a given cell background in development or disease. Ligand-dependent Cre expression and ligand-inducible Cre function provide a means, in principle, to supplement the spatial control conferred by lineage-specific promoters alone. This is more than just an a priori issue for cardiac muscle cells; Cre transgenes driven by the myosin light chain-2v or MHC upstream control regions both were reported to be sufficiently expressed during embryogenesis to induce efficient recombination in ventricular myocardium before birth.^{5 6} In the latter case, this is ascribed to early transcription of MHC in presumptive ventricular cells, at the linear heart tube stage.²² Indeed, using the ROSA26 Cre reporter line, we have found recombination frequencies of up to 90% can be elicited by MHC-Cre in both atrial and ventricular myocytes at mid- gestation (V. Gaussin and M.D. Schneider, unpublished results, 2000).

Consequently, we have tested the hypothesis that conditional activation of a Cre fusion protein might enable heart-specific recombination to be deferred until postnatal life, using a truncated LBD derived from the human progesterone receptor. Using the MHC promoter, CrePR1 expression was directed to cardiac myocytes in vivo. Immunolocalization confirmed that CrePR1 was diffusely expressed in the absence of RU486 and was preferentially translocated to cardiac myocyte nuclei in the presence of this synthetic antiprogestin. Cardiac-specific expression of CrePR1 was found to mediate RU486-inducible recombination selectively in myocardium. The relative absence of recombination under basal conditions 1 or more weeks after birth stands in marked contrast to results obtained by ourselves and others using constitutively functional Cre protein with the identical MHC promoter⁶ (V. Gaussin and M.D. Schneider, unpublished results, 2000), or published results using myosin light chain-2v–Cre⁵ ; both trigger high-level recombination in the ventricle before birth, as cited above.

To our knowledge, the present report is also the first direct evidence for ligand-dependent translocation of Cre, using the progesterone receptor LBD. This is not a foregone conclusion, because full-length wild-type progesterone receptor is constitutively nuclear, unlike the glucocorticoid receptor.²³ However, additional mechanisms may exist for the activation of chimeric proteins via this domain.²⁴ Conceptually and logistically, ligand-dependent activation of Cre may be advantageous, by comparison with ligand-dependent expression, given the need for just 1 exogenous protein, not 2, for the inducible recombination event.²⁵ Spontaneous recombination in the absence of RU486 specifically required CrePR1 (recombination does not occur between the paired loxP sites otherwise) and was related to the respective levels of Cre expression across 3 independent transgenic lines. Hence, even lower levels of expression might be optimal than seen in our lowest copy line. Immunolocation does not preclude the presence of CrePR1 in the nucleus at low basal levels, constitutively, below the threshold for detection by this method. Several potential mechanisms may contribute to recombination in the absence of RU486. Despite the proven selectivity of this mutated LBD for synthetic progesterone antagonists,^{7 26} resulting from deletion of the 42–amino acid C-terminal domain, weak interaction with endogenous steroid hormones can be envisioned. Even though the major nuclear targeting motif for the progesterone receptor is disrupted (amino acids 638 to 641, in the hinge domain), secondary karyophilic signals exist within the LBD itself.²³ In addition, CrePR1 contains an engineered nuclear localization signal, previously shown to potentiate the action of Cre,²⁷ which had no adverse effect on "leaky" recombination by the fusion protein, at least under the conditions of transient transfection.⁷ Because gene recombination results in an irreversible somatic mutation, cells containing a recombined gene will accumulate over time, making inducible deletion inherently more susceptible to leak than is inducible transcription of exogenous genes. This would be especially true for cells such as cardiac myocytes, with a long life span, and this has been also seen with neurons.^{8 9}

The potential utility for posttranslational activation of Cre in the cardiovascular system is most obvious where phenotypes are desirable after birth and the developmental regulation of tissue-specific promoters is insufficient to achieve this purpose. Apart from improving the spatial and temporal control of endogenous genes’ deletion, conditional activity of Cre can supplement conditional expression for more precise control of Cre-dependent transgenes where recombination is used for irreversible induction (by removing a loxP-flanked stop cassette³ ) or for irreversible down-regulation (by excision). In all 3 circumstances, ligand-activated recombination may also be useful to help establish gene function at different times or at different extents of deletion. The latter is analogous to the use of chimeric mice with differing ratios of wild-type and mutant cells,²⁸ but is more readily workable as the need for ongoing creation of new blastocysts is obviated. Moreover, progressive, time-dependent, mosaic recombination might be more relevant than uniform deletion in creating genetic models of chronic, acquired disease such as heart failure, in which marked tissue heterogeneity is known to exist at the protein level. Illustrations of this include ion channels such as connexin-43,²⁹ as well as sarcomeric proteins.³⁰ In addition, genomic heterogeneity within an individual (somatic mosaicism) is characteristic of the cardiac involvement in certain triplet-repeat disorders.³¹ To minimize spontaneous recombination, especially at later ages, lower baseline expression, conditional expression, or further refinement of the Cre fusion protein are avenues to be pursued. However, even in this present form, posttranslational activation of Cre permits highly efficient inducible recombination in cardiac muscle, or progressive recombination in the absence of the drug, bypassing potential embryonic and perinatal effects on the heart.

	Acknowledgments

 
This work was supported by grants from the NIH and by the M.D. Anderson Foundation Professorship (to M.D.S.). We gratefully acknowledge the indicated authors for reagents; B. Boerwinkle, J. Pike, and F. Ervin for technical assistance; and R. Roberts for encouragement and support.

	Footnotes

 
Original received January 18, 2001; revision received January 31, 2001; accepted January 31, 2001.

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