Download Inducible Gene Targeting in Postnatal Myocardium by Cardiac

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
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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 ␤-galactosidase (␤-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. (Circ Res. 2001;88:
587-592.)
Key Words: Cre recombinase 䡲 genetics 䡲 progesterone receptor 䡲 transgenic mice
G
ene 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 38kDa 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 – 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.7–11
These chimeric proteins become selectively active on binding
a synthetic ligand, eg, RU486 or tamoxifen in preference to
endogenous progesterone and estrogen, respectively. An at-
Original received January 18, 2001; revision received January 31, 2001; accepted January 31, 2001.
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 [email protected]
© 2001 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
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tractive extension of this technology would confine Cremediated 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
Transgenic Mice
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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/␮L, 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 9⫻105 cells
per 35-mm well for gene transfer. Transfection was carried out using
lipofectamine, 1.0 ␮g of the loxP-tagged CAG-CATZ reporter,15 0.5
␮g of an SV40-driven luciferase reporter gene to correct for
transfection efficiency, and 2.0 ␮g 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 ␮L, 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 ␮g/mL PMSF, 50 ␮mol/L leupeptin, and 50 ␮mol/L
aprotinin. Cell lysates (50 ␮g 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, ␤-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 ␮g/mL DAPI, and cardiac myocytes were
identified using MF20 antibody to sarcomeric MHC (1 ␮g/mL;
University of Iowa Hybridoma Bank) followed by Texas Red–
conjugated antibody against mouse IgG (Molecular Probes).14
To quantify Cre-dependent recombination using ␤-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-␮m 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 ␤-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 multiplecomparison test, using a significance level of P⬍0.05.
Results
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.7–9 As initial validation studies, cultured ventricular
myocytes were cotransfected with the Cre-dependent reporter
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Inducible Gene Targeting in Postnatal Myocardium
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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 ␮g of
pCAG-CATZ and 0.5 ␮g 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 recombinationdependent 690-bp fragment.
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 ␤-actin promoter15; downstream of
CAT is the Escherichia coli ␤-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 ␤-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.
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). Transgenepositive 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
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).
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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).
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).
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.
RU486 Induces Cardiac-Specific Recombination in
␣MHC-CrePR1ⴛROSA26 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
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 promoterdriven neomycin phosphotransferase gene. Bottom, PCR analysis of DNA from representative mice at the age of 6 weeks, indicating amplification of the recombination-dependent fragment.
␤-Casein was used as a positive control. H indicates heart; Lu,
lung; Li, liver; S, spleen; and Q, skeletal muscle (quadriceps).
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-CrePR1⫻Rosa26 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.
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
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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 ␮m. Left, Representative data. Right, Mean results.
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.
Discussion
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
591
found recombination frequencies of up to 90% can be elicited by
␣MHC-Cre in both atrial and ventricular myocytes at midgestation (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
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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 tissuespecific 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 Credependent 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.
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Inducible Gene Targeting in Postnatal Myocardium by Cardiac-Specific Expression of a
Hormone-Activated Cre Fusion Protein
Tetsuo Minamino, Vinciane Gaussin, Francesco J. DeMayo and Michael D. Schneider
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
Circ Res. 2001;88:587-592
doi: 10.1161/01.RES.88.6.587
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