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作者：Antoine Ouvrard-Pascaud, Stefania Puttini, Yannis Sainte-Marie, Rafika Athman, Virginie Fontaine, Françoise Cluzeaud, Nicolette Farman, Marie-Edith Rafestin-Oblin, Marcel Blot-Chabaud, and Frederic Jaisser作者单位：1 Institut National de la Santé et de la Recherche Médicale U47 Federative Institute of Research 0 Bichat Medical School, 75870 Paris Cedex 18; and 2 Centre National de la Recherche Scientifique UMR 14 Institut Curie, 75006 Paris, France
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【摘要】$ O0 O; B) A+ u* P
The renal collecting duct plays a key role in control of ion and fluid homeostasis. Genes encoding for ion transporters, hormone receptors, or regulatory proteins specifically expressed in the collecting duct are mutated in several genetic diseases with altered blood pressure. Suitable cellular models expressing genes in a conditional way should represent attractive systems for structure-function analyses and generation of appropriate physiopathological models of related diseases. However, generation of such systems remains laborious and quite inefficient. We adapted and improved a conditional Cre-lox-inducible system in the highly differentiated aldosterone-sensitive rat cortical collecting duct (RCCD2) cell line. The inducible MerCreMer recombinase allowed tight control and high levels of transgene expression, whereas flanking a selection marker with two loxP sites strongly improved the selection procedure. We have used this system to conditionally express an enhanced green fluorescent protein-tagged human mineralocorticoid receptor. In the future, this will allow structure-function analyses as well as mineralocorticoid receptor trafficking studies in these epithelial cells, which retain the features of the native collecting duct. Improvements in the conditional Cre-lox expression system have potentially wide applications in other epithelial or nonepithelial cell lines. ) Z8 l- Z; l$ @/ s& B
【关键词】 Cre recombinase rat cortical collecting duct cells mineralocorticoid receptor aldosterone
& N5 v3 t! E. N% ^1 D- g HIGHLY DIFFERENTIATED MAMMALIAN cell lines that retain the main features of the native cells represent a powerful tool to study its physiological properties ex vivo ( 30 ). Cellular models may also help us understand complex and specific mechanisms, such as hormonal and drug responses. Constitutive expression of an exogenous protein can lead to various deleterious effects. Chronic cellular adaptation may result in phenotypic compensations, complicating the precise analysis of the relationship between exogenous gene expression and functional consequences. If the expressed protein is cytotoxic, later analysis and survival of the model are compromised. To alleviate these problems, it is necessary to turn off expression of the transgene until it is required for the experimental protocol. Inducible systems offer this possibility ( 17 ). Moreover, they allow control experiments in the same cellular model, i.e., with the same genetic alteration, with or without expression of the transgene. We previously adapted a functional tetracycline-inducible (Tet-On) system in an epithelial rat cortical collecting duct (RCCD) cell line ( 26 ). Although this strategy was well adapted to the temporal control of transgene expression, the study of cytotoxic proteins is not possible, because the transgene is expressed during the selection period. We have therefore developed an alternative strategy that uses the Cre-lox conditional system with an efficient protocol to select cell clones with conditional transgene expression.) D; v5 h; V! f6 w- {( s7 W
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Cre is a 38.5-kDa recombinase encoded by the bacteriophage P1, which catalyzes site-specific recombination between two loxP sites, consisting of two 13-bp inverted repeats separated by a spacer; Cre can excise DNA flanked by two loxP sites positioned head-to-tail ( Fig. 1 ) ( 28 ). The Cre coding sequence is fused to the ligand-binding domain (LBD) of the estrogen receptor (ER), so that the activity of the modified Cre recombinase can be controlled over time ( 22 ). In the absence of its ligand (17 -estradiol), the inducible Cre ER remains in the cytoplasm, where it is proposed to be bound to the heat shock protein complex ( 5, 13, 16 ). In the presence of 17 -estradiol, Cre ER is translocated to the nucleus, where it can exert its enzymatic activity ( 5 ). However, the 17 -estradiol ligand has many other endogenous targets, inducing unwanted stimulation of endogenous gene expression. To improve the specificity of the response to ligand administration, several inducible Cre recombinases have been developed that use mutated steroid receptor LBDs that are sensitive to synthetic ligand ( 4, 10, 11, 16, 18, 19 ).2 i5 ]: f1 X% s( Z1 z' X
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Fig. 1. Outline of the Cre-lox-inducible system, a bigenic system. The 1st construct allows expression of a fusion protein (Pr), including Cre and a mutated ligand-binding domain (LBD) of the estrogen receptor (ER), which is insensitive to the natural ligand 17 -estradiol but activated by the synthetic ligands tamoxifen and 4-hydroxytamoxifen (4-OH-Tam). Cre can excise a DNA sequence flanked by two 34-bp loxP recognition sites positioned head- to-tail (so-called floxed DNA). The 2nd construct harbors 2 distinct transgenes ( A and B ) to be differentially expressed. Two loxP sites flank transgene A. In the absence of 4-OH-Tam, the inducible Cre recombinase remains inactive and is sequestered in the cytoplasm, resulting in appropriate transcription of the floxed transgene A, whereas transgene B is not expressed. In the presence of 4-OH-Tam, the inducible Cre is translocated to the nucleus, where it mediates site-specific recombination and excision of the floxed cassette, allowing transcription of transgene B.$ b N8 Z4 M& z9 n1 k% ^$ |7 [
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Conditional overexpression of the transgene of interest can be achieved using a specifically designed construct that includes two independent transcription units under control of a common strong promoter. Expression is dependent on removal of a stop cassette located between the promoter and the transgene of interest. Because the stop cassette is flanked by two loxP sites, it can be excised once the site-specific Cre recombinase is activated. This stop cassette consists of tandems of stop codons or coding sequences (e.g., selection marker and reporter gene) followed by polyadenylation [poly(A)] sequences.
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In this study, we have used two different inducible Cre recombinases to develop a conditional system for transgene expression in a renal epithelial cell line originating from the CCD, the target of aldosterone. We compared the CreER T2 recombinase developed by Feil et al. ( 11 ), in which Cre is fused to a mutated LBD (G400V/M543A/L544A) of the human ER, with the MerCreMer recombinase developed by Zhang and al. ( 32 ), in which Cre is fused at NH 2 and COOH termini to a mutated binding domain (G525A) of the murine ER. Although these mutated ER LBDs do not bind any more 17 -estradiol, the synthetic ligands tamoxifen and 4-hydroxytamoxifen behave as agonists ( 11, 32 ). Moreover, we have used this system to express conditionally an enhanced green fluorescent protein (eGFP)-tagged human mineralocorticoid receptor (hMR) in the aldosterone-responsive RCCD2 cell line ( 8 ).
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MATERIALS AND METHODS' e# r2 _! a2 ?% c2 e" E
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* i2 z' _! G3 _3 IRCCD2 cells were cultured on petri dishes that had been coated with rat type I collagen, as previously described ( 8 ). Cells were cultured in a defined medium that contained 1:1 Ham's F-12-DMEM with 14 mM NaHCO 3, 20 mM HEPES buffer (pH 7.4), 10 U/ml penicillin-streptomycin, 2 mM glutamine, 5 µg/ml insulin, 50 nM dexamethasone, 5 µg/ml transferrin, 50 nM sodium selenite, 50 nM triiodothyronine, 10 µg/ml epidermal growth factor, and 2% FCS (Invitrogen, Groningen, The Netherlands), hereafter referred to as complete medium. When required, cells were cultured in a medium without dexamethasone, triiodothyronine, and insulin and with 2% charcoal-stripped FCS, i.e., without steroids, hereafter called steroidfree medium.
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0 Y! s+ b8 v1 \- K0 R* q, tPlasmid Constructs
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) b w' V8 ~( }Cytomegalovirus (CMV)-CreER T2 -internal ribosome entry site (IRES)-neomycin (Neo) construct. CreER T2 cDNA was isolated after Eco RI digestion from the pCreER T2 plasmid ( 11 ) and subcloned in the Eco RI site of the pIRES-Neo vector (Clontech, Palo Alto, CA).
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" _2 w8 K( T6 g2 `9 }7 h* A8 e& uCMV-MerCreMer-IRES-Neo construct. MerCreMer cDNA was isolated after Hin dIII digestion from the pAN-MerCreMer plasmid ( 32 ) and subcloned after blunt ligation in the Eco RI site of the pIRES-Neo vector (Clontech).
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CMV-loxP-chloramphenicol acetyl transferase (CAT)-loxP-LacZ construct. The loxP-CAT-loxP-LacZ cassette was isolated after Xba I digestion from pCAG-loxP-CAT-loxP ( 1 ) and subcloned in the Xba I site of a pcDNA 3.1 hygromycin (Hygro) 5.6 vector (Invitrogen) previously digested with Kpn I/ Not I and blunt ligated on itself to excise part of the polylinker site.
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4 R) K, M1 A6 ~. v' }2 _$ tCMV-loxP-Hygro-loxP-LacZ construct. The hygromycin resistance gene cDNA was isolated after Sma I/ Pvu II digestions from the pcDNA 3.1 Hygro 5.6 vector (Invitrogen) and subcloned after blunt ligation in the Xho I site of the CMV-loxP-CAT-loxP-LacZ construct.
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CMV-loxP-Hygro-loxP-eGFPhMR construct. We first modified the phMR3750 plasmid (kindly provided by R. M. Evans) by incorporating a Bgl II site after ligation of the GGCCATAGATCTAT oligonucleotide, subcloned as a dimer in the Xma III site of phMR3750. The hMR cDNA was isolated after Bgl II/ Hin dIII digestions from this modified phMR3750 and subcloned in the Bgl II/ Hin dIII sites of the peGFP-C1 vector (Clontech). The eGFP-hMR fusion cDNA was isolated after Age I/ Mlu I digestions from the phMR-eGFP-C1 vector and subcloned after blunt ligation in the Bam HI site of the CMV-loxP-Hygro-loxP-LacZ construct.% U) m- J8 _- W# F0 ~1 d5 H- A! {
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Stable Transfections of RCCD2 Cells With CMV-CreER T2 -IRESNeo or CMV-MerCreMer-IRES-Neo Construct' m, T5 P) c. D" y1 r' Q
5 O+ u' f, ?: I! e$ `CMV-CreER T2 -IRES-Neo and CMV-MerCreMer-IRES-Neo constructs were linearized after Dra I digestion. Native RCCD2 cells ( passage 8 ) were seeded at 2.7 x 10 6 cells in six-well plates 24 h before transfection. For each well, 1 µg of the linearized constructs and 4 µl of Reagent Plus (Invitrogen) were incubated at room temperature in 100 µl of Optimem (Invitrogen) for 15 min and then added to 3 µl of Lipofectamine (Invitrogen) previously diluted in 100 µl of Optimem, incubated again at room temperature for 15 min, and added with 800 µl of Optimem. Cells were incubated for 4 h with this mixture before it was replaced with complete medium. At 2 days after transfection, cells were incubated with 400 µg/ml G418 (Invitrogen). Cells were then seeded at very low density, and stably transfected cells were selected with 400 µg/ml G418. G418-resistant cells were isolated, amplified, and characterized.
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+ t4 r1 S: H; A% FX-Gal Staining Assay9 M6 A8 E! N) Z: q
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CIN and mIN clones, which constitutively express CreER T2 - and MerCreMer-inducible recombinases, respectively, were transiently transfected with 1 µg of the circular CMV-loxP-CAT-loxP-LacZ or CMV-loxP-Hygro-loxP-LacZ construct (see above). The cells were stimulated for 48 h with 100 nM 4-hydroxytamoxifen, rinsed with PBS buffer, fixed for 5 min at 4°C with 2% paraformaldehyde in PBS buffer, rinsed again three times with PBS buffer, and then incubated for 12 h at 37°C with 1 mg/ml 5-bromo-4-chloro-3-indolyl- - D -galactopyranoside (X-Gal; Sigma-Aldrich, St. Quentin Fallavier, France) in X-Gal buffer [2 mM MgCl 2, 5 mM K 4 Fe(CN) 6 ·3 H 2 O, 5 mM K 3 Fe(CN) 6, and 150 mM NaCl in 1 x PBS buffer].( t9 y& P' q# _
; }5 P x. k1 y" S KStable Transfections of RCCD2 Cells Expressing Inducible Cre Recombinases With CMV-loxP-Hygro-loxP-LacZ or CMV-loxPHygro-loxP-eGFPhMR Constructs0 {* O1 ~8 O: p3 X9 @8 P
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The CIN 22 clone constitutively expressing the CreER T2 -inducible recombinase was stably transfected with the CMV-loxP-Hygro-loxPLacZ construct. The mIN 8 clone, constitutively expressing the MerCreMer-inducible recombinase, was stably transfected with the CMV-loxP-Hygro-loxP-LacZ or CMV-loxP-Hygro-loxP-eGFPhMR construct. Stable transfections were performed as described above, except cells were selected in the presence of 300 µg/ml hygromycin (Invitrogen). Hygromycin-resistant cells were isolated, amplified, and characterized. CMV-loxP-Hygro-loxP-LacZ and CMV-loxP-HygroloxP-eGFPhMR constructs were linearized after Nru I/ Stu I digestions, which allow excision of the hygromycin resistance gene from the original pcDNA 3.1 Hygro 5.6 vector.
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-Galactosidase Activity Assay; k; I3 z G3 ~+ h' j
6 q3 K* e* M/ }1 p& Z; KMeasurements were performed on RCCD2 subclones expressing the CreER T2 or MerCreMer Cre recombinase and the reporter transgene CMV-loxP-Hygro-loxP-LacZ. Cells were seeded to be confluent when harvested for enzymatic dosage. For dose-response experiments, cells were stimulated or not stimulated for 48 h with 4-hydroxytamoxifen. A time course experiment was performed in clone mIN 8.28 LacZ after addition of 10 nM 4-hydroxytamoxifen. After 48 h, the medium was discarded and the cells were lysed in 250 µl of lysis buffer for -galactosidase assay ( 27 ). Measurements are achieved by spectrophotometry (wavelength = 420 nm). Values are means ± SE from triplicate experiments.' A, l3 _2 X' \% ~
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Northern Blot Experiments& s: I& M" ~3 \7 L
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Cells were cultured on collagen-coated filters (Costar 3412, Corning). Cells were seeded at 3 x 10 6 cells per filter. After 96 h, complete medium was replaced with steroid-free medium. After 16 h, 1 nM aldosterone was added or not added. After 24 h, total RNAs were extracted from cells with TRIzol reagent (Invitrogen) according to the manufacturer's recommendations. Total RNAs (20 µg) were run on a 0.8% denaturating glyoxal-agarose gel and blotted onto nylon membranes (Positive TM Membrane, Q.BIOgene, Montreal, PQ, Canada). Membranes were then hybridized with random [ - 32 P]dCTP-labeled probes encoding for rat -subunit of epithelial Na channel ( -ENaC: 590 bp, nt 2185-2775), serum and glucocorticoid-induced kinase (Sgk: 671 bp, nt 314-985), and glyceraldehyde phosphate dehydrogenase (GAPDH: 851 bp, nt 20-871) as control of RNA amounts. Specific hybridization signals were quantified by measuring radioactivity with a Packard Instant Imager. Values, expressed in arbitrary units, correspond to the ratios of -ENaC or Sgk to GAPDH and are means ± SE from four experiments.
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+ H0 c. ] Y9 _$ cElectrophysiological Studies$ t& F4 W5 O: c1 s q, v
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Short-circuit current ( I sc, µA/cm 2 ) was measured in mIN 8.11 eGFP-hMR cells cultured on collagen-coated Snapwell filters (Costar 3407, Corning), as described previously ( 3a ). Briefly, Snapwell filters were mounted into a voltage-clamp system, and cells were bathed on each side with 8 ml of medium thermostated at 37°C in 95% O 2 -5% CO 2. I sc was measured by clamping transepithelial voltage to 0 mV for 1 s. To study the effect of amiloride on I sc, mIN 8.11 eGFP-hMR cells were grown on Snapwell filters for 96 h in complete medium. I sc was measured before ( time 0 ) and after 15 min of exposure to 10 µM amiloride. Alternatively, to study the effect of aldosterone, cells grown on Snapwell filters were incubated overnight in a minimum medium containing 1:1 Ham's F-12-DMEM with 14 mM NaHCO 3, 2 mM glutamine, 10 U/ml penicillin-streptomycin, and 20 mM HEPES (pH 7.4). Aldosterone (10 nM) was then added or not added to the cells, and incubation continued for 24 h in minimum medium.5 J' \1 [/ f- S' M" |' j5 r+ M
; H: S" b7 g) LDetermination of Transcriptional Activity' \: h( i( s) f. l
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Transcriptional activity of eGFP-hMR was determined as previously described ( 9 ) in cells transiently transfected as described above with 5 µg of pFC31-luciferase vector [a reporter plasmid containing the mouse mammary tumor virus (MMTV) promoter upstream of the luciferase gene] ( 14 ) and 1 µg of simian virus-40- -galactosidase vector (Promega, Madison, WI). mIN 8.11 eGFP-hMR cells were seeded at 15 x 10 5 cells in six-well plates. After 24 h, complete medium was replaced with steroid-free medium. 4-Hydroxytamoxifen (10 nM) was added or not added 16 h later. Transient transfection was performed 24 h later. Aldosterone (1 nM) or 100 nM spironolactone was added or not added to the cells 24 h after transfection in steroid-free medium without 4-hydroxytamoxifen, and incubation continued for 24 h. Cell extracts were then prepared and assayed for luciferase ( 7 ) and -galactosidase activities ( 27 ). To standardize for transfection efficiency, the relative light units obtained in the luciferase assay were divided by the optical density obtained in the -galactosidase assay. Values are means ± SE from triplicate experiments.- K2 y- Q6 t9 x P8 y( x
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Confocal Microscopy
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Experiments were performed on clone mIN 8.11 eGFP-hMR. Cells were seeded at 2.5 x 10 5 cells in a chambered coverglass system (Lab-Tek, Christchurch, New Zealand) in complete medium. Once cells settled and started to divide, medium was replaced with steroidfree medium. After 24 h, cells were stimulated or not stimulated with 150 nM 4-hydroxytamoxifen for 48 h. Confocal microscopy analyses were performed to determine the subcellular localization of the eGFPhMR fusion protein before and after addition of 1 nM aldosterone for 30 min at 37°C in the presence of 5% CO 2.
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- L. H5 ]$ {5 Z8 Q0 c# tStatistical Comparison of Groups* M7 p4 Z9 x* v8 o; F( O
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Values are means ± SE, and comparison between means was made using unpaired Student's t -test.
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RESULTS# o6 j2 u4 Q8 `4 R$ |0 K3 x
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Establishment and Improvement of the Cre-lox-Inducible System in the RCCD2 Cell Line D) ^" h+ z F5 y
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A CMV-CreER T2 -IRES-Neo construct ( Fig. 2 A ) was stably transfected in the RCCD2 cells to obtain constitutive expression of CreER T2 recombinase. The IRES sequence allows transcription of a bicistronic mRNA ( 15 ), resulting in expression of CreER T2 fusion protein and the Neo selection marker under control of the same promoter. To assess the ability of the G418-selected RCCD2 clones to mediate Cre-inducible recombination, transient transfections with a CMV-loxP-CAT-loxPLacZ reporter construct ( Fig. 2 A ) were followed by 48 h of stimulation with 100 nM 4-hydroxytamoxifen. Because of the presence of a poly(A) site after the CAT cDNA, the LacZ reporter gene cannot be expressed until the CAT-poly(A) sequence is excised. When activated, the inducible CreER T2 recombinase will catalyze excision of the floxed CAT-poly(A) cassette, allowing expression of the LacZ gene. As estimated by X-Gal staining, 9 of 12 (75%) G418-resistant clones displayed inducible expression of LacZ (data not shown). However, a leak of -galactosidase activity was observed for each clone in the absence of 4-hydroxytamoxifen induction. A clone (hereafter called CIN 22 for CreER T2 -IRES-Neo 22) was chosen for further evaluation, because it showed a better ratio for expression of LacZ in the presence or absence of 4-hydroxytamoxifen. Because leakiness could be due to the particular conditions of transient transfection, in which a large quantity of the reporter construct is present as extrachromosomal, nonintegrated DNA, the CIN 22 clone was stably transfected with the reporter construct.
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! j7 e: R" O7 S5 g& C+ RFig. 2. Establishment of a Cre-lox-inducible system using CreER T2 recombinase. A : rat cortical collecting duct (RCCD2) cells were stably transfected with a cytomegalovirus (CMV) - CreER T2 -internal ribosome entry site sequence (IRES)-neomycin construct, which allows constitutive expression of CreER T2 recombinase, and functionally selected after transient transfection with a CMV-loxP-chloramphenicol acetyl transferase (CAT)-loxP-LacZ reporter construct. Selected clones were then stably transfected with a CMV-loxPhygromycin (Hygro)-loxP-LacZ reporter construct in which the hygromycin marker allows clonal selection of the transfected cells. B : -galactosidase ( -gal) activity in 3 double-transgenic subclones (CIN 22.10, CIN 22.16, and CIN 22.17) after stimulation (or no stimulation) with 10 nM 4-OH-Tam compared with background levels measured in native RCCD2 cells and singly transfected CIN 22 cells. C : dose-response curve of LacZ expression in 4-OH-Tam-induced cells. -Galactosidase activity, measured in the CIN 22.10 LacZ subclone after stimulation for 48 h with 0-100 nM 4-OH-Tam, was maximal for 10 nM 4-OH-Tam. Values are means ± SE ( n = 3).$ Y+ Q- @9 O/ t4 n% C3 t$ l1 t3 `
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As reported by others, we previously experienced difficulty in obtaining doubly transfected clones with a strong expression of the relevant conditional construct. To improve the process of selection, the CMV-loxP-CAT-loxP-LacZ construct was modified, with the hygromycin selection marker introduced between the loxP sites ( Fig. 2 A ). This strategy allows expression of the selection marker and the LacZ reporter gene under control of the same promoter. In the absence of 4-hydroxytamoxifen, the hygromycin resistance gene is expressed. After stimulation of CreER T2 recombinase in the presence of 4-hydroxytamoxifen and excision of the floxed cassette, the LacZ reporter gene is expressed. If the hygromycin selection pressure is maintained during cell expansion, cultured cells that display spontaneous Cre recombinase activity in the absence of stimulation with 4-hydroxytamoxifen will die, because they will no longer resist hygromycin. Therefore, this approach allows the induction of expression of the gene of interest only after excision of the selection marker.
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* c4 y [" t/ D5 w! i2 zThe CMV-loxP-Hygro-loxP-LacZ reporter construct was stably transfected in the CIN 22 clone. Seventeen G418/hygromycin-resistant subclones were isolated. Three (23%) of these subclones were positive as assessed by an X-Gal staining assay after 48 h of stimulation with 100 nM 4-hydroxytamoxifen (data not shown). However, expression of LacZ remained undetectable in the other subclones, indicating that activity of the CMV promoter at the integration site is sufficient for expression of the hygromycin resistance gene at a threshold level for selection of subclones but is not sufficient to allow detectable expression of LacZ. Alternatively, the random integration of the reporter construct may have resulted in an incomplete LacZ coding sequence. -Galactosidase activity was estimated on the three positive subclones compared with the native RCCD2 cells and the singly transfected CIN 22 cells ( Fig. 2 B ). In cells stimulated or not stimulated with 100 nM 4-hydroxytamoxifen for 48 h, responsiveness and expression of LacZ were high compared with background levels measured in the native RCCD2 cells. However, a leak of -galactosidase activity was present in the absence of 4-hydroxytamoxifen, indicating spontaneous activity of the CreER T2 -inducible recombinase in these cells. Subclone 10 (hereafter called CIN 22.10 LacZ), which displayed the highest -galactosidase activity, was chosen for further characterization. A dose-response curve was performed on clone CIN 22.10 LacZ, and the plateau of -galactosidase activity was reached after 48 h of stimulation with 5 nM 4-hydroxytamoxifen ( Fig. 2 C ). This result is in agreement with those observed in other cell types using inducible recombinases in which Cre is fused to a mutated LBD at its COOH-terminal end ( 13, 18, 19, 32 ).
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$ R: R; i7 Q5 `& @) o1 j- vTo overcome leakiness of the CreER T2, we used another inducible Cre recombinase that has been previously reported to be tighter, i.e., MerCreMer, a fusion protein between Cre and two mutated binding domains of the murine ER (mER) ( 32 ). A CMV-MerCreMer-IRES-Neo construct ( Fig. 3 A ) was stably transfected in the RCCD2 cell line. Functional expression of the MerCreMer recombinase was estimated in G418-resistant clones. After transient transfection with the CMV-loxP-HygroloxP-LacZ reporter transgene, cells were stimulated or not stimulated with 100 nM 4-hydroxytamoxifen for 48 h. X-Gal staining showed that three of seven clones (43%) displayed conditional expression of LacZ (data not shown). A clone (hereafter called mIN 8 for MerCreMer-IRES-Neo subclone 8 ) was chosen for further characterization, because it displayed the highest staining intensity after 4-hydroxytamoxifen induction. Next we stably transfected the clone mIN 8 with the CMV-loxP-Hygro-loxP-LacZ reporter construct. Thirty double-transgenic subclones were obtained after hygromycin selection, amplified, and stimulated or not stimulated with 100 nM 4-hydroxytamoxifen for 48 h. X-Gal staining indicated that 12 subclones (40%) displayed conditional expression of LacZ (data not shown). No leak of -galactosidase activity was observed in these subclones in the absence of stimulation of the MerCreMer-inducible recombinase with 4-hydroxytamoxifen. Measurements of -galactosidase activity in subclone 28 (hereafter called mIN 8.28 LacZ) were performed compared with the native RCCD2 cells and the singly transfected mIN 8 cells. In the absence of 4-hydroxytamoxifen stimulation, -galactosidase activity in mIN 8.28 LacZ cells was similar to that in native RCCD2 cells, i.e., background level, whereas it was largely induced after stimulation ( Fig. 3 B ). Furthermore, no leak of -galactosidase activity could be observed in the absence of induction of the MerCreMer recombinase by 4-hydroxytamoxifen, even when the selection pressure was turned off, in contrast to RCCD2 cells expressing the CreER T2 recombinase ( Fig. 2 C ). A dose-response curve was obtained after stimulation for 48 h with different amounts of 4-hydroxytamoxifen. The plateau of -galactosidase activity was reached at a low concentration (10 nM) of 4-hydroxytamoxifen ( Fig. 3 C ). A time course of induction of the -galactosidase activity was performed. Cells were stimulated for 0-100 h in the presence of 10 nM 4-hydroxytamoxifen. The maximal enzyme activity was observed within 50 h after the beginning of induction ( Fig. 3 D ). These results indicate that the MerCreMer recombinase appears to be more suitable than the CreER T2 recombinase to establish a conditional expression system in immortalized cell lines and that the use of the MerCreMer chimeric recombinase is an effective strategy to control transgene expression in the highly differentiated RCCD2 epithelial cell line.8 @ u% K1 g1 T# c
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Fig. 3. Establishment of a Cre-lox-inducible system using the MerCreMer recombinase. A : RCCD2 cells were stably transfected with a CMV-MerCreMer-IRES-neomycin construct, which allows constitutive expression of MerCreMer recombinase. G418-resistant clones were then stably transfected with the CMV-loxP-Hygro-loxP-LacZ reporter construct and selected in the presence of hygromycin. In the presence of 4-OH-Tam, site-specific recombination allowed excision of the floxed selection marker and expression of LacZ. B : -galactosidase activity in the doubly transfected subclone mIN 8.28 after stimulation (or no stimulation) with 10 nM 4-OH-Tam compared with background levels in native RCCD2 cells and singly transfected mIN 8 cells. C : dose-response curve of 4-OH-Tam-induced LacZ expression. -Galactosidase activities were measured in the mIN 8.28 LacZ subclone after stimulation with 1-100 nM 4-OH-Tam. LacZ activity was maximal for 10 nM 4-OH-Tam. D : time course of LacZ expression. -Galactosidase activities were measured in the mIN 8.28 LacZ subclone after stimulation with 10 nM 4-OH-Tam for 0-96 h. Maximal activity was reached after 50 h. Values are means ± SE ( n = 3)./ d& v" R6 P) c& ^' V
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Expression of the eGFP-hMR Gene in RCCD2 Cells Using the Cre-lox-Inducible System
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We designed an expression construct that allows conditional expression of the wild-type hMR to study its subcellular localization and its functional properties in a cellular context close to the native target cell, i.e., the cortical collecting duct. The LacZ gene was replaced in the CMV-loxP-Hygro-loxPLacZ reporter construct by a cDNA coding for a fusion protein in which eGFP is fused to the NH 2 terminus of hMR ( Fig. 4 A ). The resulting CMV-loxP-Hygro-loxP-eGFPhMR construct was stably transfected in the mIN 8 clone, which constitutively expressed the MerCreMer-inducible recombinase. Sixteen G418/hygromycin-resistant subclones were selected, amplified, and stimulated or not stimulated with 150 nM 4-hydroxytamoxifen. Screening for the clones with the highest expression was performed by Western blot with an anti-eGFP antibody (data not shown). The clone mIN 8.11 eGFP-hMR was chosen for further characterization.
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9 ]6 N6 {6 T9 r: ]6 J0 [1 OFig. 4. Conditional expression of an enhanced green fluorescent protein (eGFP)-human mineralocorticoid receptor (hMR) fusion protein in RCCD2 cells. A : a CMV-loxP-Hygro-loxP-eGFPhMR construct was stably transfected in the mIN 8 clone, which constitutively expresses MerCreMer recombinase. In the absence of 4-OH-Tam, Cre recombinase is inactive and hygromycin resistance gene is expressed, allowing clonal selection of transfected cells. In the presence of 4-OH-Tam, Cre recombinase becomes active, and site-specific recombination allows excision of the floxed selection marker and expression of eGFP-hMR. B and C : Northern blot analyses of the -isoform of epithelial Na channel ( -ENaC) and serum and glucocorticoid-induced kinase (Sgk) expression. mIN 8.11 eGFP-hMR cells were incubated ( ) or not incubated (-) for 24 h with 1 nM aldosterone (aldo). B : total RNA was extracted and processed for analysis with specific probes for -ENaC, Sgk, and GAPDH. C : Instant Imager quantified signals. -ENaC-to-GAPDH and Sgk-to-GAPDH ratios were calculated. Results are expressed as degree of induction of -ENaC or Sgk expression in the presence of aldosterone relative to control condition. Values are means ± SE ( n = 4). * P : c7 Y& e, \8 w6 X
, M9 [2 R; K; `6 { |We analyzed whether the doubly transfected mIN 8.11 eGFP-hMR cells have retained their differentiated epithelial state during and after introduction of the Cre-lox-inducible system, i.e., after two rounds of transfection and clonal selection. This analysis was performed under conditions in which eGFP-hMR was not expressed, i.e., without 4-hydroxytamoxifen pretreatment. Expressions of -ENaC and Sgk ( Fig. 4 B ) were examined by Northern blot. Stimulation of the doubly transfected cells (clone mIN 8.11 eGFP-hMR) with 1 nM aldosterone for 24 h induced an increase in the transcripts encoding for Sgk (3.1-fold induction) and -ENaC (1.6-fold induction; Fig. 4 C ). To evaluate the electrophysiological properties of the doubly transfected cells in the absence of transgene expression, amiloride-sensitive I sc was measured in doubly transfected cells in the absence of 4-hydroxytamoxifen induction. After 15 min of exposure to 10 µM amiloride, an inhibitor of ENaC, a 40% decrease in I sc was observed: from 2.1 ± 0.1 µA/cm 2 at time 0 to 1.2 ± 0.2 µA/cm 2 at 15 min ( n = 3, P
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Transactivation activity of the eGFP-hMR was examined in transfection assays using a reporter plasmid containing the MMTV promoter upstream of the luciferase gene ( Fig. 4 D ). Without 4-hydroxytamoxifen stimulation, mIN 8.11 eGFPhMR cells do not respond to the MR ligand aldosterone or the MR antagonist spironolactone. In cells stimulated with 4-hydroxytamoxifen, 1 nM aldosterone induced an increase in luciferase activity; incubation with 1 nM aldosterone and 100 nM spironolactone prevented the aldosterone-induced MR transactivation activity ( Fig. 4 D ). Control experiments using a promoterless luciferase gene (pGL2-Basic, Promega) showed that aldosterone had no effect on luciferase activity, confirming aldosterone selectivity for the MMTV promoter (data not shown).
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/ M. C1 x1 v/ j6 [; m5 VConfocal microscopy analyses showed that, in the absence of stimulation with 4-hydroxytamoxifen, no eGFP fluorescence was observed in the cells, reflecting the tight control of eGFPhMR expression in this model ( Fig. 4 E ). In the presence of 4-hydroxytamoxifen, but in the absence of aldosterone, eGFPhMR was expressed and distributed over cytoplasm and nucleus ( Fig. 4 E ). After 30 min of incubation with 1 nM aldosterone, the ligand receptor was translocated into the nucleus, where the fluorescence was concentrated ( Fig. 4 E ).
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DISCUSSION4 P: q8 h9 U5 i- c
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Our goal was to define the best strategy to obtain conditional gene expression in the highly differentiated RCCD2 cells. Using site-specific recombination mediated by Cre recombinase, we established a renal epithelial cell line suitable for reliable and inducible gene expression. We developed a rapid and efficient selection procedure resulting in a conditional strategy that allows high transgene expression with no background expression in the absence of induction. This strategy allows temporal control of gene expression when required for the experimental protocol, avoiding adaptive or deleterious effects of the transgene expression during the establishment of the transgenic cell clones or during the control phase of experiments.
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6 F4 v" X% T3 v1 R4 M7 `. J4 [3 CAs shown in the present study, 4-hydroxytamoxifen-dependent gene recombination mediated by the CreER T2 -inducible recombinase described by Feil et al. ( 11 ) is highly efficient in the RCCD2 epithelial cell line. However, we observed a major leakiness with time, resulting in basal expression of the reporter constructs in the absence of 4-hydroxytamoxifen stimulation. Leakiness in the system is a major drawback, because every recombination event is irreversible; therefore, even a low recombinase activity will result in a high accumulation of recombined target gene with time and cell passages, except if an efficient counterselection procedure is applied, as described in our study with a selection marker flanked by loxP sites. It has been suggested that basal activity may result from proteolysis of the Cre-LBD fusion protein or from cryptic splicing of the corresponding mRNA ( 31, 32 ). Both events could result in constitutively active Cre molecules when the LBD moiety is absent. Zhang and co-workers ( 32 ) reasoned that a duplication of the LBD by fusion at the NH 2 - and COOH-terminal ends of Cre recombinase would result in less background activity, because protease activity will result in truncated proteins with no Cre activity (in the case of COOH-terminal truncation) or maintained ligand-dependent activity (in the case of NH 2 -terminal truncation). Using a mutated G525R murine ER LBD (mER), they generated the so-called MerCreMer-inducible Cre recombinase used in the present study; its activity is tightly dependent on 4-hydroxytamoxifen. Indeed, when the MerCreMer recombinase was used, none of the double-transgenic RCCD2 cells selected for G418/hygromycin resistance were leaky, and most of these cells displayed high expression on 4-hydroxytamoxifen induction. Importantly, improvement in design of the conditional expression constructs, including the selection marker between the loxP sites in the stop cassette, avoids the screening of nonexpressing clones and favors selection of highly expressing clones.
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These inducible Cre recombinases have been initially developed to allow temporal control of gene modification, such as gene inactivation in vivo in transgenic mice ( 23 ). However, they have been only rarely used for conditional gene expression in immortalized cell lines, mainly because of the difficulties encountered in establishing a reliable and tightly controlled system ex vivo. Most of these studies have been performed to test and improve the functional properties of the developed inducible Cre recombinase in nondifferentiated cell lines such as CV1 cells derived from green monkey fibroblast ( 18, 19 ), F9 mouse embryonic carcinoma cells ( 4, 16, 22 ), or the MEF 5/5 murine embryonic fibroblast line ( 29 ). Using transgenic cells established from the J558mb-I myeloma of B lymphocytes that constitutively express the MerCreMer recombinase, Zhang et al. ( 33 ) reported conditional expression of the micromolar heavy chain of immunoglobulins. Introduction of the Cre-loxinducible system in the RCCD2 cells should be useful in the study of physiological consequences of transgene expressions in a highly differentiated epithelial cell line ex vivo.
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Using this tight system, we have conditionally expressed an eGFP-hMR fusion protein in RCCD2 cells, a cell line that recapitulates several functional properties of the native cortical collecting duct, including ion transport, high transepithelial resistance, and aldosterone sensitivity ( 8 ). Previous experiments performed in our laboratory revealed that, for unknown reasons, it has not been possible to overexpress constitutively the MR in these differentiated cells in the absence of an inducible strategy (data not shown). The Cre-lox-inducible system allowed us to circumvent this problem, because hMR can be expressed only during the experimental procedure and not during cell culture, preventing a hypothetically deleterious effect on the cells. Here we show that the fusion protein eGFP-hMR expressed in RCCD2 cells using the Cre-lox system retains functional properties of the native cell line. Aldosterone-induced transactivation activity is maintained in the presence of eGFP at the NH 2 terminus of the hMR and is inhibited by the antagonist spironolactone. Thus conditional eGFP-hMR expression appears to be a good alternative to transient expression of the fusion protein to analyze nucleocytoplasmic trafficking of MR in living cells. In the mIN 8.11 eGFPhMR cells, the level of expression of eGFP-hMR appears sufficient to allow experiments to analyze the structurefunction relationship of MR as well as aldosterone-induced genes in cortical collecting duct cells. Experiments are in progress in the laboratory to study the influence of eGFPhMR overexpression, i.e., with 4-hydroxytamoxifen pretreatment, on transepithelial Na transport and aldosteroneinduced genes.% l r, q# N y# c) O8 v4 j7 ~
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In conclusion, we have developed a powerful conditional expression strategy that can be applied to other cell lines of interest. Using this approach, we were able to establish stable collecting duct cell lines with conditional overexpression of proteins, including an eGFP-tagged MR. The Cre-lox conditional system appears to be useful to generate appropriate cellular models for physiological studies.
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# p& ^* a) m: j. KACKNOWLEDGMENTS
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" I' A7 p8 f' _! F9 t( p9 ]% zWe thank J. Miyazaki, D. Metzger, M. Reth, and R. Evans for providing the plasmid constructs. We are grateful to A. T. Beggah for help in generation of the conditional expression constructs." c9 u* r: L, N7 \
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GRANTS
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2 V3 m O! x& U$ ]- g) WA. Ouvrard-Pascaud and S. Puttini were supported by fellowships from the Ministère de la Recherche et de la Technologie and the Fondation pour la Recherche Médicale. This work was supported by the Institut National de la Santé et de la Recherche Médicale and an Action Concertée Incitative Grant from the Ministère de la Recherche et de la Technologie.
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Address for reprint requests and other correspondence: F. Jaisser, Equipe Avenir, Collège de France, 11 Place Marcelin Berthelot, 75231 Paris, France (E-mail: frederic.jaisser{at}college-de-france.fr 0 E$ J. N9 R1 K8 N9 ^! g) f1 M3 k
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