SPECIAL COMMUNICATION
Tetracycline-inducible gene expression in cultured rat renal CD cells and in intact CD from transgenic mice

Stefania Puttini, Ahmed T. Beggah, Antoine Ouvrard-Pascaud, Christine Legris, Marcel Blot-Chabaud, Nicolette Farman, and Frederic Jaisser

Institut National de la Santé et de la Recherche Médicale U478, Federative Institute of Research 02, Bichat Medical School, 75870 Paris Cedex 18, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger (AE1) are expressed in the CD. Most of these transporters are regulated by hormones and compounds such as vasopressin, aldosterone, or adrenergics, which facilitate a fine control of the final composition of the urine. It has been shown in the recent years that mutation(s) of these transporters are responsible for several inherited tubular disorders. Pseudohypoaldosteronism has been linked to mutations of ENaC subunits (4), autosomal dominant distal renal tubular acidosis is associated with mutations of AE1 (19), whereas Bartter's syndrome has been related to mutations in the genes encoding for the Na+-K+-2Cl- 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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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).


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 1.   Outline of the tetracycline-inducible system ("Tet system"). The Tet system, is a bigenic system, allowing inducible expression of a gene of interest. A first construct, a so-called "transactivator construct" (rtTA), allows expression of a fusion protein between a strong transcription factor (VP16 from Herpes simplex virus) and the Tet repressor, isolated from Escherichia coli. In the presence of an exogenous ligand, i.e., tetracycline antibiotic or its derivatives such as doxycycline (Dox), rtTA is able to interact with the Tet operon sequences linked to a minimal cytomegalovirus (CMV) promoter (pmin) in the responder construct. Transcription of the transgene is turned on only in the presence of Dox (+Dox) but is turned off in its absence (-Dox).

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.


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 2.   Establishment of a RCCD1-Tet-On subclone. A: RCCD1 collecting duct cells were first stably transfected with the CMV-rtTA transactivator construct. This allowed constitutive expression of the rtTA transactivator in the RCCD1 cells, resulting in RCCD1/Tet-On cells. Transient transfection with the pminLacZ (pBI-3) bidirectional construct allows inducible expression of the lacZ reporter gene under the control of the Tet promoter. A multicloning site (MCS) allows the insertion of a second cDNA in the pBI-3 construct if needed. B: the RCCD1/Tet-On B5 subclone expresses lacZ in the presence (+), but not in the absence (-), of Dox when cells are transiently transfected with pBI-3, but not with the control vector pBI-4. Relative beta -galactidose (gal) activity is normalized to the internal control (luciferase). C: dose-response curve of lacZ gene expression. beta -Gal activity was measured in RCCD1/Tet-On cells (B5 subclone) transiently transfected with pBI-3 and cultured in the presence of various concentrations of Dox for 48 h. Values are means ± SE (n = 3).

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.


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 3.   The bicistronic construct pPuroIRES-SEAP allows the identification of Tet- inducible cell clones. A: RCCD1-Tet-On cells (B5 subclone) were stably transfected with the pPuroIRES-SEAP responder construct, where IRES refers of an internal ribosome entry site sequence, SEAP is secreted alkaline phosphatase, and Puro is the puromycine resistance gene. This resulted in the inducible expression of a bicistronic mRNA, allowing translation of Puro and the SEAP reporter gene. B: after puromycine selection in the presence of Dox, SEAP activity was analyzed in various subclones in the presence (+) or absence (-) of Dox. Relative SEAP activity, expressed as light units · µg protein-1 · well-1, was measured in triplicate. C: dose-response curve of SEAP expression in RCCD1-Tet-On subclone 11. Maximal expression, corresponding to the 100% of relative SEAP activity, is achieved with 5 µg/ml of Dox. D and E: time course of SEAP activity expression in RCCD1-Tet-On subclone 11 after addition (D) or removal (E) of Dox from the extracellular medium. Expression is maximal 48 h after addition of of Dox. Removal of Dox resulted in down-expression in 24 h. Values are means ± SE (n = 3).

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).


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 4.   The bidirectional construct pminHygro/LacZ is functional in RCCD1 cells. A: RCCD1-Tet-On cells (B5 subclone) were stably transfected with the pminHygro/LacZ responder construct. The bidirectional pminHygro/LacZ construct allows coordinate expression of the hygromycine resistance gene (Hygro) and the lacZ reporter gene. Note that a bicistronic mRNA could be generated by inserting a cDNA of interest into the MCS 5' to Hygro. B: after selection of various subclones in the presence of Dox and Hygro, beta -gal activity was analyzed in the presence or absence of Dox. Relative beta -gal activity, expressed as absorbance · µg protein-1 · well-1, was measured in triplicate. Values are means ± SE. C: Western blot analysis of lacZ expression in RCCD1/Tet-On 16 subclone cells treated with increasing concentrations of Dox. A major 116-kDa protein band was detected by Western blotting, using a polyclonal beta -gal antibody in cells treated with increasing concentrations of Dox, but not in the parental (Tet-On) cells nor in nontreated cells. It should be noted that the polyclonal beta -gal antibody recognized a faint, unknown 97-kDa protein band in all conditions, including RCCD1-Tet-On cells nontransfected with the pminHygro/LacZ responder construct.

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 beta -galactosidase (beta -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.

beta -Gal Assay

RCCD1-Tet-On subclones stably transfected with the pminHygro/LacZ construct were seeded at the density of 2.5 × 106 cells in six-well petri dishes and stimulated with 5 µg/ml Dox or not stimulated. After 48 h, the medium was discarded and cells were lysed in 250 µl of lysis buffer for beta -gal assay (30). Values are expressed as means ± SE from experiments conducted in triplicate.

Western Blot Analysis

Cells were lysed with 250 µl of the same lysis buffer as for the beta -gal assay. Protein content was determined by the method of Lowry (24). Proteins were separated in denaturating SDS-7.5% polyacrylamide gel (50 µg/lane) and then blotted onto polyvinylidene fluoride sheets (Hybond-P, Amersham Pharmacia Biotech). Blots were blocked overnight at 4°C with 5% nonfat dry milk in 20 mmol/l Tris · HCl, 137 mmol/l NaCl, and 0.1% Tween 20 (TBS-T). The membrane was then incubated with a polyclonal antibody against beta -gal (Capell, ICN Biomedicals; dilution 1:1,000) for 1 h at 4°C. After several washes (3 × 15 min in TBS-T), the membrane was incubated with the second anti-rabbit antibody (Santa Cruz Biotechnology) at a dilution of 1:10,000 for 1 h at 4°C. After several washes (3 × 15 min in TBS-T), beta -gal protein was detected by chemiluminescent reaction (ECL+ kit, Amersham) followed by exposure of the membrane to BioMax MR-1 film (Kodak).

Inducible 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 beta -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 beta -gal was performed on the same sections, using a polyclonal anti-beta -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).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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). beta -Gal activity was determined in the RCCD1-Tet-On subclones 2 days after transient transfection, in the presence or absence of 5 µg/ml Dox added to the culture medium. Several subclones of RCCD1-Tet-On cells had inducible beta -gal activity. The RCCD1-Tet-On B5 subclone displayed the highest inducible expression (~30-fold) (Fig. 2B). A dose-response curve was established with the use of increasing concentrations of Dox (Fig. 2C). The results indicated that, when tested after transient expression of a reporter construct, maximal activity of the system was achieved with ~0.5-1 µg/ml Dox. Therefore, the RCCD1-Tet-On B5 subclone was chosen for all subsequent experiments.

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 Omega /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 both beta -gal and a hygromycine selection marker in the RCCD1-Tet-On cell line (Fig. 4A). Twenty-three clones were isolated after stable transfection, and selection was performed in the presence of Dox and hygromycine. Seven clones expressed the selection marker in a Dox-dependent manner, and four were used for further characterization. All selected clones exhibited inducible lacZ expression at high levels, although a few had background expression in the absence of Dox (Fig. 4B). Expression was inducible but leaky in the absence of Dox in clone 9, whereas expression was highly inducible and not leaky in clone 16, for example. Western blot analysis performed with RCCD1/Tet-On subclone 16 showed a dose-dependent expression of the lacZ gene (Fig. 4C), whereas no expression was observed in the absence of Dox or in parental cells. Dose-response curves indicated that this construct allows similar responsiveness to Dox to that with the pPuroIRES-SEAP construct (data not shown).

Inducible Expression of the lacZ Reporter Gene in the Collecting Ducts From Transgenic Mice

We next analyzed the expression of beta -gal in double-transgenic mice obtained from intercrosses between CMV-rtTA mice described by Kirstner et al. (L3 strain) (20) and the lacZ responder line (LacZ17) (Fig. 5A). Intercross of this responder mouse with the transactivator mouse line allowed Dox-dependent expression of beta -gal in tissues expressing the transactivator (Beggah AT, Puttini S, and Jaisser F, unpublished observations). Kidneys were removed from the double-transgenic mice (CMV-rtTA/LacZ17) treated with Dox for 7 days or untreated. Strong blue staining was detected in the deep cortex and in the inner medulla after 4-h incubation at RT (Fig. 5B). Interestingly, no staining was seen in the outer medulla, nor in the majority of the cortex, suggesting that neither proximal tubule cells, Henle's loop cells, nor outer medullary collecting duct cells expressed a functional rtTA transactivator. Analysis of 70-µm sections confirmed the presence of a strong expression of lacZ in tubules from the cortex and inner medulla, consistent with labeling of cortical and inner medullary collecting ducts (Fig. 5C).


View larger version (57K):
[in this window]
[in a new window]
 
Fig. 5.   The Tet system is functional in vivo in the cortical collecting duct and inner meduallry collecting duct. A: double-transgenic mice were obtained by breeding 2 independent mice strains: a transactivator mouse strain (L3 strain), in which the expression of the rtTA transactivator is under the control of the constitutive CMV promoter, and a responder strain (L17 strain), transgenic for a bidirectional responder construct similar to those described in Figure 2A. B: double-transgenic mice resulting from crossbreeding the CMV-rtTA mice and the lacZ reporter mice were injected daily with (+Dox) or without (-Dox) for 7 days. Note that the lacZ reporter gene (blue) was only expressed when mice were treated with Dox (+ Dox). C: illustration of intrarenal 5-bromo-4-chloro-3-indoyl beta -D-galactopyranoside (X-Gal) staining in the kidneys of double-transgenic mice treated with Dox for 7 days. A 70-µm vibratome section was obtained from the X-Gal-stained kidney slices. Note the presence of intense blue staining in some tubules from the cortex and papilla. D: heterocellular expression of nuclear beta -gal was observed in microdissected inner medullary collecting duct, as revealed by X-Gal staining. E: colocalization of beta -gal (red) with Dolichos biflorus agglutinin (green), a lectin that specifically labels collecting ducts, further confirmed that the lacZ reporter gene was expressed in collecting ducts.

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 beta -gal varied among adjacent cells (heterocellular expression) (Fig. 5D). The nuclear localization of the blue reaction product demonstrated that the beta -gal activity originated from the transgene and was not due to endogenous lysosomal beta -gal-like activity. Colocalization of beta -gal with DBA, a lectin that specifically labels collecting ducts, further confirmed that the beta -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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 (beta -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 beta -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 alpha -ENaC subunit (13) and a mutated beta -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 alpha -EnaC subunit knockout (13) and by the increase in renal potassium reabsorption when a mutated H+-K+-ATPase beta -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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Baron, U, Freundlieb S, Gossen M, and Bujard H. Co-regulation of two gene activities by tetracycline via a bidirectional promoter. Nucleic Acids Res 23: 3605-3606, 1995[ISI][Medline].

2.   Bens, M, Vallet V, Cluzeaud F, Pascual-Letallec L, Kahn A, Rafestin-Oblin ME, Rossier BC, and Vandewalle A. Corticosteroid-dependent sodium transport in a novel immortalized mouse collecting duct principal cell line. J Am Soc Nephrol 10: 923-934, 1999[Abstract/Free Full Text].

3.   Blot-Chabaud, M, Laplace M, Cluzeaud F, Capurro C, Cassingena R, Vandewalle A, Farman N, and Bonvalet JP. Characteristics of a rat cortical collecting duct cell line that maintains high transepithelial resistance. Kidney Int 50: 367-376, 1996[ISI][Medline].

4.   Chang, SS, Grunder S, Hanukoglu A, Rosler A, Mathew PM, Hanukoglu I, Schild L, Lu Y, Shimkets RA, Nelson-Williams C, Rossier BC, and Lifton RP. Mutations in subunits of the epithelial sodium channel cause salt wasting with hyperkalaemic acidosis, pseudohypoaldosteronism type 1. Nat Genet 12: 248-253, 1996[ISI][Medline].

5.   Courtois-Coutry, N, Roush D, Rajendran V, McCarthy JB, Geibel J, Kashgarian M, and Caplan MJ. A tyrosine-based signal targets H/K-ATPase to a regulated compartment and is required for the cessation of gastric acid secretion. Cell 90: 501-510, 1997[ISI][Medline].

6.   Coutry, N, Farman N, Bonvalet JP, and Blot-Chabaud M. Synergistic action of vasopressin and aldosterone on basolateral Na(+)-K(+)-ATPase in the cortical collecting duct. J Membr Biol 145: 99-106, 1995[ISI][Medline].

7.   De Wet, JR, Wood KV, DeLuca M, Helinski DR, and Subramani S. Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol 7: 725-737, 1987[ISI][Medline].

8.   Furth, PA, St Onge L, Boger H, Gruss P, Gossen M, Kistner A, Bujard H, and Hennighausen L. Temporal control of gene expression in transgenic mice by a tetracycline-responsive promoter. Proc Natl Acad Sci USA 91: 9302-9306, 1994[Abstract/Free Full Text].

9.   Gossen, M, and Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 89: 5547-5551, 1992[Abstract].

10.   Gossen, M, Freundlieb S, Bender G, Muller G, Hillen W, and Bujard H. Transcriptional activation by tetracyclines in mammalian cells. Science 268: 1766-1769, 1995[ISI][Medline].

11.   Hirt, RP, Poulain-Godefroy O, Billotte J, Kraehenbuhl JP, and Fasel N. Highly inducible synthesis of heterologous proteins in epithelial cells carrying a glucocorticoid-responsive vector. Gene 111: 199-206, 1992[ISI][Medline].

12.   Hogan, B, Beddington R, Costantini F, and Lacy E. Manipulating the Mouse Embryo. Plainview, NY: Cold Spring Harbor Laboratory, 1994.

13.   Hummler, E, Barker P, Talbot C, Wang Q, Verdumo C, Grubb B, Gatzy J, Burnier M, Horisberger JD, Beermann F, Boucher R, and Rossier BC. A mouse model for the renal salt-wasting syndrome pseudohypoaldosteronism. Proc Natl Acad Sci USA 94: 11710-11715, 1997[Abstract/Free Full Text].

14.   Igarashi, P, Shashikant CS, Thomson RB, Whyte DA, Liu-Chen S, Ruddle FH, and Aronson PS. Ksp-cadherin gene promoter. II. Kidney-specific activity in transgenic mice. Am J Physiol Renal Physiol 277: F599-F610, 1999[Abstract/Free Full Text].

15.   Jaisser, F. Inducible gene expression and gene modification in transgenic mice. J Am Soc Nephrol 11: S95-S100, 2000.

16.   Jaisser, F, and Beggah AT. Transgenic models in renal tubular physiology. Exp Nephrol 6: 438-446, 1998[ISI][Medline].

17.   Jou, TS, and Nelson WJ. Effects of regulated expression of mutant RhoA and Rac1 small GTPases on the development of epithelial (MDCK) cell polarity. J Cell Biol 142: 85-100, 1998[Abstract/Free Full Text].

18.   Jou, TS, Schneeberger EE, and Nelson WJ. Structural and functional regulation of tight junctions by RhoA and Rac1 small GTPases. J Cell Biol 142: 101-115, 1998[Abstract/Free Full Text].

19.   Karet, FE, Finberg KE, Nayir A, Bakkaloglu A, Ozen S, Hulton SA, Sanjad SA, Al-Sabban EA, Medina JF, and Lifton RP. Localization of a gene for autosomal recessive distal renal tubular acidosis with normal hearing (rdRTA2) to 7q33-34. Am J Hum Genet 65: 1656-1665, 1999[ISI][Medline].

20.   Kistner, A, Gossen M, Zimmermann F, Jerecic J, Ullmer C, Lubbert H, and Bujard H. Doxycycline-mediated quantitative and tissue-specific control of gene expression in transgenic mice. Proc Nat Acad Sci USA 93: 10933-10938, 1996[Abstract/Free Full Text].

21.   Kitamura, M. Creation of a reversible on/off system for site-specific in vivo control of exogenous gene activity in the renal glomerulus. Proc Nat Acad Sci USA 93: 7387-7391, 1996[Abstract/Free Full Text].

22.   Kothary, R, Barton SC, Franz T, Norris ML, Hettle S, and Surani MA. Unusual cell specific expression of a major human cytomegalovirus immediate early gene promoter-lacZ hybrid gene in transgenic mouse embryos. Mech Dev 35: 25-31, 1991[ISI][Medline].

23.   Kress, C, Vogels R, De Graaff W, Bonnerot C, Meijlink F, Nicolas JF, and Deschamps J. Hox-2.3 upstream sequences mediate lacZ expression in intermediate mesoderm derivatives of transgenic mice. Development 109: 775-786, 1990[Abstract].

24.   Lowry, ON, Rosebrough NJ, Farr AL, and Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-275, 1951[Free Full Text].

25.   Miquerol, L, Cluzeaud F, Porteu A, Alexandre Y, Vandewalle A, and Kahn A. Tissue specificity of L-pyruvate kinase transgenes results from the combinatorial effect of proximal promoter and distal activator regions. Gene Expr 5: 315-330, 1996[ISI][Medline].

26.   Montoliu, L, Chavez S, and Vidal M. Variegation associated with lacZ in transgenic animals: a warning note. Transgenic Res 9: 237-239, 2000[ISI][Medline].

27.   Nelson, RD, Stricklett P, Gustafson C, Stevens A, Ausiello D, Brown D, and Kohan DE. Expression of an AQP2 Cre recombinase transgene in kidney and male reproductive system of transgenic mice. Am J Physiol Cell Physiol 275: C216-C226, 1998[Abstract/Free Full Text].

28.   Pognonec, P, and Chambard JC. A reliable way of obtaining stable inducible clones. Nucleic Acids Res 26: 3443-3444, 1998[Abstract/Free Full Text].

29.   Quabius, ES, Murer H, and Biber J. Expression of proximal tubular Na-Pi and Na-SO4 cotransporters in MDCK and LLC-PK1 cells by transfection. Am J Physiol Renal Fluid Electrolyte Physiol 270: F220-F228, 1996[Abstract/Free Full Text].

30.   Sambrook, J, Fritsch EF, and Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor, 1989.

31.   Simon, DB, Bindra RS, Mansfield TA, Nelson-Williams C, Mendonca E, Stone R, Schurman S, Nayir A, Alpay H, Bakkaloglu A, Rodriguez-Soriano J, Morales JM, Sanjad SA, Taylor CM, Pilz D, Brem A, Trachtman H, Griswold W, Richard GA, John E, and Lifton RP. Mutations in the chloride channel gene, CLCNKB, cause Bartter's syndrome type III. Nature Genet 17: 171-178, 1997[ISI][Medline].

32.   Simon, DB, Karet FE, Hamdan JM, DiPietro A, Sanjad SA, and Lifton RP. Bartter's syndrome, hypokalaemic alkalosis with hypercalciuria, is caused by mutations in the Na-K-2Cl cotransporter NKCC2. Nature Genet 13: 183-188, 1996[ISI][Medline].

33.   Simon, DB, Karet FE, Rodriguez-Soriano J, Hamdan JH, DiPietro A, Trachtman H, Sanjad SA, and Lifton RP. Genetic heterogeneity of Bartter's syndrome revealed by mutations in the K+ channel, ROMK. Nature Genet 14: 152-156, 1996[ISI][Medline].

34.   Srinivas, S, Goldberg MR, Watanabe T, D'Agati V, al-Awqati Q, and Costantini F. Expression of green fluorescent protein in the ureteric bud of transgenic mice: a new tool for the analysis of ureteric bud morphogenesis. Dev Genet 24: 241-251, 1999[ISI][Medline].

35.   Srinivas, S, Wu Z, Chen CM, D'Agati V, and Costantini F. Dominant effects of RET receptor misexpression and ligand-independent RET signaling on ureteric bud development. Development 126: 1375-1386, 1999[Abstract/Free Full Text].

36.   Stricklett, PK, Nelson RD, and Kohan DE. Targeting collecting tubules using the aquaporin-2 promoter. Exp Nephrol 7: 67-74, 1999[ISI][Medline].

37.   Wang, T, Courtois-Coutry N, Giebisch G, and Caplan MJ. A tyrosine based signal regulates H-K-ATPase-mediated potassium reabsorption in the kidney. Am J Physiol Renal Physiol 275: F818-F826, 1998[Abstract/Free Full Text].


Am J Physiol Renal Fluid Electrolyte Physiol 281(6):F1164-F1172
0363-6127/01 $5.00 Copyright © 2001 the American Physiological Society