Institut National de la Santé et de la Recherche Médicale U478, Federative Institute of Research 02, Bichat Medical School, 75870 Paris Cedex 18, France
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ABSTRACT |
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First published August
30, 2001; 10.1152/ajprenal.00360.2000.The renal collecting duct
(CD) plays a key role in the control of ion and fluid homeostasis.
Several genetic diseases that involve mutations in genes encoding for
ion transporters or hormone receptors specifically expressed in CD have
been described. Suitable cellular or transgenic animal models
expressing such mutated genes in an inducible manner should represent
attractive systems for structure-function relationship analyses and the
generation of appropriate physiopathological models of related
diseases. Our first goal was to develop a CD cell line that allows
inducible gene expression using the tetracycline-inducible system
(Tet-On). We designed several strategies aimed at the development of a
tight and highly inducible system in RCCD1 cells, a rat cortical collecting duct (CCD) cell line exhibiting several properties of the
native CCD. Analysis of reporter gene expression demonstrated that the
Tet-On system is suitable for inducible gene expression in these cells.
In a second step, we have tested whether transgenic Tet-On mice
expressing the tetracycline transactivator under the control of the
human cytomegalovirus promoter were suitable for inducible gene
expression in tubule epithelial cells. The results indicate that, in
vivo, the inducible expression of the lacZ reporter gene
appeared to be restricted to the CD. This particular strain of
transgenic mice may therefore be useful for the expression of genes of
interest in an inducible manner in the collecting duct.
transgenic; inducible systems; conditional systems; kidney; physiopathology
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INTRODUCTION |
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THE RENAL
CORTICAL COLLECTING duct (CCD) plays a major role in fluid,
electrolyte, and acid-base homeostasis. Several ion transporters and
exchangers, including the epithelial Na+ channel (ENaC),
the ATP-sensitive K+ channel (ROMK), the
H+-K+-ATPases, and the
Cl/HCO
transporter, the ROMK
potassium channel, or the ClC-K1 chloride channel
(31-33). Cellular as well as animal models expressing
the mutated transporters or ion channels responsible for these genetic diseases would represent powerful tools to analyze electrolyte, acid-base, and fluid homeostasis disorders (16).
Constitutive transgenes may, however, cause lethality or produce
undesirable adaptive responses that may impair the analysis of the
animal phenotypes.
Bujard et al. (9, 10) have developed a system called the "tetracycline-inducible system" (Tet system), which allows time-dependent control of gene expression ex vivo in cultured cells, as well as spatiotemporal control of gene expression in vivo in transgenic mice (8, 20). The Tet system is based on the use of a fusion protein between a strong transcription factor (VP16 from Herpes simplex virus) and the Tet repressor isolated from Echerichia coli. This fusion transcription factor (rtTA) is able to interact with the Tet operon sequences (Tet-O) linked to a minimal cytomegalovirus (CMV) promoter. Transcription of the transgene is turned on only in the presence of an exogenous ligand, Tet, or its derivative, doxycycline (Dox), but is turned off in its absence. This Tet-On system, therefore, allows precise control of the timing of expression of the transgene of interest. Furthermore, the transactivation is ligand dose dependent, and thus the level of expression of the gene of interest may also be controlled. Finally, withdrawal of the exogenous ligand allows the shutdown of expression and a return to basal experimental conditions. These features distinguish this system from the more commonly used constitutive-promoter systems, wherein the expression of the gene is not under quantitative or temporal control.
The goal of this study was to establish a conditional expression system ex vivo by using a differentiated cell line originating from the rat cortical collecting duct and to develop a similar system for in vivo inducible gene expression along the nephron. We adapted the Tet system to cultured RCCD1 cells, a rat cortical collecting duct cell line (3). We have also designed constructs that alleviate the screening of the inducible cell clones, a problem usually encountered with commercial Tet vectors. In addition, we have shown that the Tet-On system is functional in vivo in collecting ducts from kidneys of transgenic mice and that the CMV promoter restricts the inducible expression of a reporter gene to cortical and inner medullary collecting ducts.
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MATERIALS AND METHODS |
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General Strategy
The Tet system allows inducible expression of a transgene, the coding sequence of which is placed under the control of the Tet-inducible promoter. In the Tet-On system, transactivation of the Tet-inducible promoter is mediated by the reverse Tet transactivator (rtTA) in the presence of Dox only. The Tet system, therefore, requires the presence of two transgenic constructs in target cells: a transactivator construct and a responder construct (Fig. 1).
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Ex Vivo Experiments
We first stably transfected the rat RCCD1 cell line (3) with a transactivator construct. Stable transfectants, called RCCD1-Tet-On cells, were then functionally characterized by transient transfection with a responder construct, allowing Dox-dependent expression of a reporter gene. The best transactivator cell line was selected in view of high expression of the responder construct in the presence of Dox and absence of expression without Dox. In a second step, a responder construct, allowing inducible expression of the transgene of interest, was stably transfected in these cells.In Vivo Experiments
Two strains of transgenic mice were required. The first strain comprised transactivator mice that are transgenic for a transactivator construct, allowing either ubiquitous or tissue-specific expression of the transactivator, depending on the promoter used to control its expression. The second strain was composed of responder mice that are transgenic for a responder construct, allowing its inducible expression in a Dox-dependent manner. In the present study, double-transgenic mice were obtained by mating CMV-rtTA transactivator mice and lacZ responder mice to allow the presence of the two transgenic constructs in the target cells. Expression of the responder construct was then tested in mice with or without Dox treatment.Plasmid Constructs
Transactivator construct. The CMV-rtTA construct was kindly provided by H. Bujard (10). It allows constitutive expression of rtTA under the control of the CMV-IE promoter. The plasmid also contains a neomycine resistance gene under the control of the SV40 constitutive promoter.
First responder construct.
For transient transfection assays, the circular bidirectional pminLacZ
construct (pBI-3; kindly provided by H. Bujard) (1) was
transfected in RCCD1-Tet-On cell clones (Fig.
2A). This construct allowed
inducible expression of the lacZ reporter gene under the control of the
Tet promoter.
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Second responder construct.
A HindIII-HindIII puromycine resistance gene
(Puro) was isolated from plasmid pT-Puro and subcloned in the
HindIII site of the pTis vector (both plasmids kindly
provided by P. Pognonec) (28). This resulted in
the pPuroIRES-SEAP construct (Fig.
3A). This bicistronic
construct includes a puromycine selection marker (Puro), an internal
ribosome entry site sequence (IRES), followed by a cDNA encoding for
the secreted alkaline phosphatase (SEAP) (Fig. 3A). SEAP is
secreted in the extracellular medium, allowing convenient measurement
of reporter gene activity in living cells. Because of the presence of
IRES in pPuroIRES-SEAP, only one mRNA is produced but two proteins are
translated. The pPuroIRES-SEAP construct was linearized with
FspI for stable transfection in RCCD1-Tet-On cells. Stable
transfectants were selected in the presence of 5 µg/ml Dox and 2 µg/ml puromycine.
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Third responder construct.
The IRES-hygromycine resistance gene (Hygro) was excised from
pCMV-IRES-Hygro (Clontech) with SpeI and XbaI.
After Klenow treatment, the blunted insert was ligated into the blunted
SalI site of the pminLacZ construct (pBI-3). This resulted
in the bidirectional pminHygro/LacZ construct (Fig.
4A). This pminHygro/LacZ
construct allows coordinated expression of both Hygro and the lacZ
reporter gene. The presence of an IRES in front of Hygro allows the
5'-insertion of a cDNA of interest if needed, resulting in a
bicistronic and bidirectional construct. The pminHygro/LacZ construct
was linearized with BsaI for stable transfection in
RCCD1-Tet-On cells (B5 subclone).
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Establishment of Stably Transfected RCCD1 Cells With the Transactivator Construct
Native RCCD1 cells (3) were seeded at the density of 2.5 × 106 cells in six-well plates 24 h before transfection. One microgram of linearized CMV-rtTA construct and 4 µl of Reagent plus (Life Technologies) were incubated in 100 µl of OPTIMEM (Life Technologies) for 15 min, then added to 3 µl of lipofectamine previously diluted in 100 µl of OPTIMEM and incubated at RT for 15 min. Eight hundred microliters of OPTIMEM were added to DNA complexes. The mixture was then added to the cells. The medium containing the DNA complexes was replaced with fresh medium after 4-h incubation. Two days after transfection, cells were seeded and stably transfected cells were then selected with 400 µg/ml geneticine (Life Technologies). Geneticine-resistant RCCD1-Tet-On cells were isolated, amplified, and characterized.RCCD1-Tet-On subclones were functionally characterized by transient
transfection, as described above, with a lacZ reporter construct
(pBI-3) or a control vector (pBI-4) (1). Transfection efficiency was normalized with the use of the constitutive expression vector RSV40-luciferase (7). Two days after transfection,
the cells were lysed, and -galactosidase (
-gal) activity was
measured and normalized to the internal luciferase control.
Establishment of Stably Transfected RCCD1-Tet-On Cells With the Reporter Constructs
RCCD1-Tet-On (B5 subclone) cells were transfected with the reporter constructs as described above. Twenty-four hours after transfection, 5 µg/ml Dox (Sigma) was added for 48 h. Cells were then seeded, and stably transfected cells were selected in the presence of 5 µg/ml Dox with 2 µg/ml puromycine (for transfection with the pPuroIRES-SEAP construct; Sigma) or 400 µg/ml hygromycine (for transfection with the pminHygro/LacZ construct; Life Technologies). Puro- or Hygro-resistant clones were then isolated, amplified, and characterized.SEAP Assay
RCCD1-Tet-On subclones stably transfected with the pPuroIRES-SEAP construct were seeded at the density of 2.5 × 106 cells in six-well petri dishes, and 5 µg/ml Dox was or was not added. After 48 h, 250 µl of medium were transferred to a microcentrifuge tube for SEAP assay (Great Escape SEAP Detection kit, Clontech). Extracellular medium was removed, and cells were lysed in 250 µl lysis buffer for protein quantification. SEAP activity was related to the protein content in each well. Values are given as means ± SE from experiments conducted in triplicate.-Gal Assay
Western Blot Analysis
Cells were lysed with 250 µl of the same lysis buffer as for theInducible Expression of the lacZ Reporter Gene in the Kidney of Transgenic Mice
The transactivator mouse line CMV-rtTA (L3 strain) was kindly provided by H. Bujard. This strain has been previously described by Kirstner et al. (20). It allows Dox-dependent transgene expression in several organs, including brain, heart, lung, liver, and kidneys (20). To define which cell type functionally expresses the Tet system in the kidney, CMV-rtTA mice were bred with lacZ responder mice established and characterized in our laboratory (Beggah AT, Puttini S, and Jaisser F, unpublished observations). The lacZ responder mice (LacZ17) are transgenic mice obtained by random integration of a bidirectional pminLacZ construct similar to the one used for characterization of RCCD1-Tet-On cells by transient transfection (see above). These responder mice allow inducible expression of lacZ in a Dox-dependent manner.5-Bromo-4-chloro-3-indoyl -D-galactopyranoside (X-Gal)
staining was examined in double-transgenic mice treated or not by daily
intraperitoneal injections of 200 µg of Dox for 7 days. The kidneys
were removed, and X-Gal staining was performed on fresh 2-mm tissue
slices soaked for 4 h at room temperature in X-Gal staining
solution (12). Kidney slices were postfixed in 4%
paraformaldehyde. Thin vibratome sections (70 µM) were then obtained
from the X-Gal-stained kidneys.
To specify the cellular origin of the X-Gal-stained cells, various
nephron segments were isolated by microdissection from Dox-treated
double-transgenic mice, as previously described (6). Isolated nephron segments were incubated at 37°C overnight in X-Gal
staining solution and photographed. Collecting ducts were also
visualized by staining 10-µm-thick cryosections with Dolichos biflorus agglutinin (DBA), a lectin that specifically labels
collecting ducts, as previously described (14).
Immunolocoalization of -gal was performed on the same sections,
using a polyclonal anti-
-gal antibody (Capell, ICN Biomedicals;
dilution 1:1,000), and revealed with a Cy3-conjugated goat-anti-rabbit
secondary antibody (dilution 1:200; Jackson Immunoresearch Laboratories).
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RESULTS |
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Selection of Stably Transfected Rat RCCD1-Tet-On Cell Lines
The rat cortical collecting duct cell line RCCD1 (3) was stably transfected with an expression construct (transactivator construct), allowing constitutive expression of rtTA (Fig. 2A). Several subclones were isolated and further characterized. Functional analysis was performed after transient transfection of a reporter construct for the Tet system, i.e., a LacZ cDNA, the expression of which was placed under the control of pminLacZ (pBI-3) (Fig. 2A).Inducible Expression of the SEAP Reporter Gene in RCCD1-Tet-On Cells With the Use of a Bicistronic Construct
The RCCD1-Tet-On B5 subclone was then used to test whether stable transfection of a reporter construct also resulted in inducible expression. Preliminary experiments using commercially available vectors indicated that stable integration of a responder construct resulted in a very high number of clones with constitutive and/or poorly inducible expression (data not shown). These results led us to improve the screening procedure, using novel constructs for the second transfection step.We designed a responder construct that allowed the coordinate expression of both reporter gene and selection marker. Stable integration of such a construct should allow, in the presence of Dox, expression of both Puro and SEAP. In this case, cells would be resistant to puromycine and secrete SEAP in the culture medium. However, in the absence of Dox, cells would become sensitive to puromycine and would not produce SEAP.
Twenty subclones stably transfected with the
pPuroIRES-SEAP construct (Fig. 3A) were isolated
in the presence of Dox. After one passage, duplicates were made
and selected in the presence of puromycine with or without Dox in the
culture medium. In the absence of Dox, all clones died when cultured in
puromycine-supplemented medium, suggesting that the expression of the
selection cassette was Dox dependent. However, among the 20 clones
tested, only 5 expressed significant Dox-dependent SEAP activity (Fig.
3B). The cellular shape (not shown) and the
electrophysiological properties of one of the RCCD1-Tet-On/SEAP
subclones (subclone 11) were similar to those of the
parental RCCD1 cells (transepithelial electrical resistance: 4,206 ± 786 /cm2; short-circuit current: 6.9 ± 0.6 µA/cm2, n = 3 ), suggesting that the
introduction of a functional Tet system into RCCD1 cells had not
altered their functional properties.
The dose-dependent response of SEAP expression, as well as the time course of expression, was established using RCCD1-Tet-On/SEAP subclone 11. Maximal SEAP expression was observed in the presence of 2 µg/ml Dox in the culture medium, as shown in Fig. 3C. The induction of SEAP expression by Dox was quite rapid, because expression was detected after 24 h and reached a plateau after 48 h. (Fig. 3D). Removal of Dox from the culture medium resulted in rapid downregulation of the expression of the reporter gene, which returned to basal levels within 24 h (Fig. 3E).
Inducible Expression of the lacZ Reporter Gene in RCCD1-Tet-On Cells With the Use of a Bidirectional Construct
Baron et al. (1) have reported another strategy using a bidirectional construct, which allows the coordinated expression of two different transgenes through the use of a single construct. To our knowledge, such a construct has not been evaluated in established cell lines (i.e., after stable transfection). We prepared a bidirectional responder construct to facilitate inducible expression of bothInducible Expression of the lacZ Reporter Gene in the Collecting Ducts From Transgenic Mice
We next analyzed the expression of
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To specify the cellular origin of the X-Gal-stained cells, various
nephron segments were isolated by microdissection from Dox-treated
double-transgenic mice (CMV-rtTA/LacZ17). X-Gal-positive cells were
observed in cortical collecting duct (not shown) and inner medullary
collecting duct (Fig. 5D) but not in proximal convoluted
tubule, cortical and medullary parts of Henle's loop, or outer
medullary collecting duct (not shown). Expression of -gal varied
among adjacent cells (heterocellular expression) (Fig. 5D).
The nuclear localization of the blue reaction product demonstrated that
the
-gal activity originated from the transgene and was not due to
endogenous lysosomal
-gal-like activity. Colocalization of
-gal
with DBA, a lectin that specifically labels collecting ducts, further
confirmed that the
-gal was expressed in collecting duct (Fig.
5E). These results indicate that, in the CMV-rtTA mice we
used, the CMV promoter had a restricted expression pattern in the
kidney, allowing specific and inducible expression of the reporter
transgene in the cortical collecting duct and inner medullary collecting duct.
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DISCUSSION |
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The development of conditional expression systems in genetically modified cells or mice is an important goal in several fields of biomedical research. It offers several advantages over constitutive expression systems and may facilitate the precise determination of the relationship between the expression of a transgene and its pathophysiological consequences (15). Inducible expression avoids undesirable transgene expression during embryonic development and reduces the potential for cellular adaptation secondary to long-term expression. Random insertion of the transgene construct may lead to other deleterious effects, such as insertional mutagenesis due to disruption of an endogenous gene. Furthermore, the number of integrated transgene copies is not controlled, and the genetic elements located in proximity to the integrated construct may affect expression of the transgene both at the quantitative and qualitative levels. This explains why independent transgenic cell lines or mice must be analyzed to avoid misinterpretation of results. In contrast, once an inducible cell clone or transgenic strain is characterized, the use of an inducible expression system permits a comparison between On- and Off-states and an analysis of the functional role of the gene product.
For these reasons, we wished to achieve, ex vivo and in vivo, inducible gene expression in the renal collecting duct. For ex vivo experiments, we chose to use the rat cortical collecting duct cell line RCCD1 to introduce the Tet-On-inducible system. These cells have many features of the native cortical collecting duct cells, i.e., high transepithelial resistance, active transepithelial Na+ transport, and response to antidiuretic hormone and isoproterenol (3). We also examined whether the mice generated in Bujard's laboratory (20) were a suitable model for in vivo inducible gene expression in collecting duct cells.
The results shown in this paper indicate that the Tet-On-inducible system is functional in cortical collecting duct cells ex vivo. This system displays several properties that make it useful for physiological studies in epithelial cell lines. Induction of expression is rapid, reaching a plateau in 48 h, and this effect is fully reversible when Dox is withdrawn. Moreover, dose-dependent expression allows a tight control of the level of expression of the foreign protein. Importantly, the results from this study show that the Tet-On system is clearly improved when a bicistronic or a bidirectional construct that includes a selection marker as well as the gene of interest is used. The inclusion of the selection marker in the inducible constructs avoids the laborious screening of nonexpressing clones. Using commercially available vectors, we (and others) have previously observed that the expression was leaky in >80% of the clones isolated after stable transfection. This may be due to the interference between the strong promoter that drives transcription of the resistance gene (used to select the stably transfected clones) and the Tet-inducible promoter. Indeed, our strategy alleviates the screening procedure, allowing rapid selection of cell clones with inducible, but not constitutive, gene expression. This strategy should be useful in establishing inducible gene expression in other renal epithelial cell types. Finally, compared with the Tet-Off system, active in the absence of Dox and repressed in its presence, the reverse Tet system (Tet-On) appears to be more convenient for physiological studies. Once the best clone is identified, it is not necessary to add Dox in the culture medium to repress transgene expression until the start of the experiment.
Other attempts have been reported in the literature to allow the
inducible expression of transgenes in renal cell lines. The synthetic
agonist dexamethasone has been used to induce transgene expression in
two kidney-derived cell lines [Madin-Darby canine kidney (MDCK) and
LLC-PK1] (29). In this case, the inducible promoter is a variant mouse mammary tumor long-terminal repeat, containing a glucocorticoid-response element (11). This
promoter has been used to express the sodium-phosphate cotransporter
and the sodium-sulfate cotransporter in a dexamethasone-dependent manner (29). The major side effect of this system is the
affinity of the ligand, dexamethasone, for the endogenous
glucocorticoid receptor, which results in undesirable activation of
transcription of glucocorticoid-responsive genes. The Tet-Off system
has been used in MDCK cells to analyze the function of proteins
involved in cell adhesion (-catenin) or in tight junctions (Rho and
Rac1 small GTPases) (17, 18). The induction was
operational, but the cell line chosen in these studies may not be
optimal for the study of collecting duct functions. Indeed, MDCK cells
have features less representative of the collecting duct than RCCD1 and
may therefore be less appropriate for studies of collecting duct
transepithelial ion transporters. Kitamura et al. (21)
have reported that the Tet-Off system is functional in renal mesangial
cells ex vivo, allowing inducible lacZ reporter gene expression.
Interestingly, these authors showed that the transfer of these
genetically modified mesangial cells into the glomeruli could be
achieved in vivo. lacZ gene expression remained inducible after in vivo
transfer, suggesting that this strategy may be relevant for cellular
therapy (21).
We have extended this work to study the functional analysis of the
Tet-On system in vivo in the kidneys of transgenic mice. Quantitative
data have been reported by Bujard et al. (8, 20), using
the Tet system in vivo. They have generated two transactivator mice
strains in which the expression of the Tet transactivator is under the
control of the strong CMV promoter, i.e., a CMV-tTA strain (Tet-Off)
and a CMV-rtTA strain (Tet-On) used in the present study.
Using a responder strain with luciferase as a reporter gene, the
authors reported a 100-fold Dox-dependent increase in luciferase
expression in whole kidney extracts (20). However, intrarenal expression was not analyzed. In the present study, we have
observed high expression of the lacZ reporter gene that is restricted
to cortical and papillary collecting ducts and was absent from the
outer medullary collecting ducts. Heterocellular expression was noted,
probably due to the well-known variegated expression of -gal in
adult tissues (26). This has also been reported in the
collecting ducts from Ksp-cadherin/LacZ transgenic mice
(14). Our results indicate that the CMV promoter has a very restricted expression pattern in the mouse kidney. We cannot exclude the fact that this is specific to the CMV-rtTA transactivator strain we have used in this study, but heterogeneous expression using
the CMV promoter in vivo has been previously reported
(22). In transgenic mice, the CMV promoter has been used
to overexpress the
-ENaC subunit (13) and a mutated
-subunit of the gastric H+-K+-ATPase
(5, 37). In both cases, expression of the transgene was
found in whole kidney extracts, but the precise intrarenal distribution
of the transgene was not defined. Functional expression in collecting
ducts was suggested by the partial rescue of the renal phenotype of the
-EnaC subunit knockout (13) and by the increase in
renal potassium reabsorption when a mutated
H+-K+-ATPase
-subunit was expressed
(37). Taken together, these results suggest that the CMV
promoter allows transgene expression in the collecting duct.
Renal-specific promoters are required to target transgene expression in specific segments of the nephron and to avoid undesirable expression in other organs. Only a few promoters allowing specific expression in collecting duct cells have been described, namely, the modified pyruvate kinase promoter (SV40-PK) (25), the aquaporin-2 (AQP2) promoter (27, 36), the Ksp-cadherin promoter (14), and the homeogene Hoxb7 promoter (23, 34, 35). The SV40-PK promoter has been shown to target the SV40 large T antigen in the thick ascending limb of the loop of Henle, the distal tubule, and the collecting duct, and to a lesser extend in proximal tubule cells (25). These mice have been used to derive highly differentiated principal collecting duct cells (2). Interestingly enough, this promoter is inducible by glucose, allowing diet-dependent expression of the transgene (25). The Ksp-cadherin promoter has been used to express the lacZ reporter gene in the collecting duct (14). The AQP2 promoter has been used to express the lacZ reporter gene or the Cre recombinase in the collecting duct (27). The AQP2-Cre mice will be useful to generate cell-specific gene inactivation in collecting duct. However, despite a large promoter region (10 kb), variegated Cre recombinase expression was reported, resulting in site-specific recombination in only 30% of the principal cells of the collecting duct (27). The Hoxb7 promoter has been used for expression of lacZ, a variant of the green fluorescent protein, and a mutant Ret protein. In the kidney, expression has been shown to be restricted to the collecting duct (23, 34, 35). The promoter is active early in embryogenesis, at day 11 when the ureteric bud is forming (23). Such an early expression may be deleterious if the functional expression of the desired protein impairs renal development, unless an inducible system is used. Taken together, one of these "collecting duct-specific" promoters should certainly be useful in efficiently targeting the renal collecting duct cells to establish a powerful inducible expression system in vivo.
In conclusion, we have reported the development of a functional Tet-On system in the RCCD1 cortical collecting duct cell line as well as in the collecting duct of transgenic mice. Such inducible systems will serve in the future to generate transgenic models for diseases related to defects of ion transporters expressed in the renal collecting duct.
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ACKNOWLEDGEMENTS |
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We thank H. Bujard and S. Freundlieb (Heidelberg, Germany) for providing the plasmid constructs of the tetracycline system and the CMV-rtTA mouse strain. Their comments and suggestions were particularly helpful at the beginning of this work. We are grateful to A. Vandewalle (Institut National de la Santé et de la Recherche Médicale U478) and C. Laing (University College, London) for helpful comments on the manuscript. We gratefully acknowledge the contributions of Sylvie Robine, who performed X-Gal staining on vibratome kidney sections, and Françoise Cluzeaud, who performed immunolocalization on kidney cryosections.
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FOOTNOTES |
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This work was supported by the Institut National de la Santé et de la Recherche Médicale and in part by grants from the Association pour la Recherche contre le Cancer. S. Puttini is supported by a fellowship from the Ministère de la Recherche et de la Technologie and the Fondation pour la Recherche Médicale and A. T. Beggah by a fellowship from the Swiss National Fund for Scientific Research.
Address for reprint requests and other correspondence: F. Jaisser, Institut National de la Santé et de la Recherche Médicale U478, Federative Institute of Research 02, Bichat Medical School, 16 rue Henri Huchard, BP 416, 75870 Paris Cedex 18, France (E-mail: jaisser{at}bichat.inserm.fr).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published August 30, 2001;10.1152/ajprenal.00360.2000
Received 31 December 2000; accepted in final form 30 July 2001.
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