Regulation of Cre Recombinase Activity by Mutated Estrogen Receptor Ligand-Binding Domains

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1 237, (1997) ARTICLE NO. RC Regulation of Cre Recombinase Activity by Mutated Estrogen Receptor Ligand-Binding Domains Robert Feil, 1 Jürgen Wagner, 2 Daniel Metzger, and Pierre Chambon 3 Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, BP 163, Illkirch-Cedex, C.U. de Strasbourg, France Received July 7, 1997 combination makes use of engineered recombinases that can be activated by ligands. Ligand-dependent re- combinases have been constructed by fusing FLP or Cre to ligand-binding domains (LBDs) of steroid recep- tors (13, 14). As these chimeric recombinases are activated by the natural ligands of the corresponding LBD, they are of limited use for applications in mice where steroids are endogenously present. Consequently, mu- tations were introduced into the LBDs of chimeric Cre recombinases that abolish the activation by natural ligands while retaining the activation by synthetic ones (15-17). We have recently demonstrated that ligandactivated site-specific recombination can be achieved by administration of tamoxifen to transgenic mice expressing Cre-ER T, a fusion protein between Cre and the G521R mutant of the human oestrogen receptor (ER) LBD (15). To improve ligand-activated site-specific recombination, we have now constructed additional Cre-ER mu- tants, and compared their recombination activities with those of Cre-ER T in F9 murine embryonal carci- noma cells. Based on their insensitivity to the natural ligand 17b-oestradiol (E2), and on their high sensitivity to the synthetic oestrogen antagonists 4-hydroxytamoxifen (OHT) and ICI 182,780 (ICI) (18), we have characterized two new ligand-dependent Cre-ER re- combinases that are of potential use to induce site- specific DNA rearrangements in mice in a spatio-tem- porally controlled manner. Ligand-dependent chimeric Cre recombinases are powerful tools to induce specific DNA rearrangements in cultured cells and in mice. We report here the construction and characterization of a series of chimeric recombinases, each consisting of Cre fused to a mu- tated human oestrogen receptor (ER) ligand-binding domain (LBD). Two new ligand-dependent recombinases which contain either the G400V/M543A/L544A or the G400V/L539A/L540A triple mutation of the human ER LBD are efficiently induced by the synthetic ER antagonists 4-hydroxytamoxifen (OHT) and ICI 182,780 (ICI), respectively, but are insensitive to 17b- oestradiol (E2). Both chimeric recombinases should be useful for efficient spatio-temporally controlled site- directed somatic mutagenesis Academic Press The possibility to introduce pre-determined somatic mutations in mice in a spatio-temporally controlled manner would greatly assist the study of mammalian gene function (1, 2). Specific DNA rearrangements can be generated by site-directed recombination using recombinases such as Cre from bacteriophage P1 (3) or FLP from Saccharomyces cerevisiae (4). At 37 C Cre is substantially more efficient than FLP and Cre is therefore recommended for applications in mice (5). Cre efficiently excises DNA flanked by directly repeated loxp recognition sites in cultured mammalian cells (6) and in mice (7, 8). Strategies for conditional gene targeting in mice based on the cell type-specific or inducible expression of constitutively active Cre have been recently described (9-12). However, up to now these systems did not allow a tight external control of induc- tion of recombination at a given time in a specific cell type (for reviews see 1, 2). An alternative approach to control site-specific re- MATERIALS AND METHODS Plasmids. To construct psgcre, the PCR product obtained by amplification of the cre gene from bacteriophage P1 DNA using the oligonucleotides 5 -CGGAATTCCACCATGTCCAATTTACTGACC- GTA-3 and 5 -GAAGATCTCTAATCGCCATCTTCCAGCAGGCG-3 was digested with EcoRI and BglII and cloned into the EcoRI-BglIIdigested psg5 expression vector (19). pcre-er(vg) and pcre- ER(GR) correspond to the previously described pcre-er (14) and 1,2 Contributed equally to this work. pcre-er T (15) expression vectors, respectively. pcre-er(gg) was 3 To whom correspondence should be addressed. Fax: (33) constructed by replacing the 252 bp HindIII-BglII fragment of pcre igbmc.u-strasbg.fr. ER(VG) with the corresponding fragment isolated from the expres X/97 $25.00 Copyright 1997 by Academic Press All rights of reproduction in any form reserved. 752

2 sion vector HEG0 (20), coding for the human ER containing a glycine Cre fused to a mutated LBD of the human ER (Fig. at amino acid 400. pcre-er(vr) was constructed by replacing the 1A). To compare the recombination activities of these 252 bp HindIII-BglII fragment of pcre-er(gr) with the correspondchimeric Cre recombinases, each expression cassette ing fragment isolated from the expression vector HE0 (21), coding for the human ER containing a valine at amino acid 400. pcre- was stably integrated into the genome of F9 cells car- ER(AA1) and pcre-er(aa2) were derived from pcre-er(vg) by site rying a lacz reporter gene (F9:LacZ cells). This reporter directed mutagenesis, using the oligonucleotides 5 -GGTGCCCCT- construct contains a loxp-flanked neomycin resistance CTATGACGCGGCGCTGGAGATGCTGGAC-3 and 5 -GACCTGCTcassette inserted into the lacz reading frame, resulting GCTGGAGGCGGCGGACGCCCACCGCCTA-3, respectively. placz(lnl) was constructed by inserting the 1.7 kb NotI fragment in a non-functional gene. After Cre-mediated excision of ppgkneoa/ls1, containing a loxp-flanked neomycin resistance of the neomycin resistance cassette one loxp site re- gene driven by the phosphoglycerate kinase (PGK) promoter, into mains in the lacz coding sequence without disrupting the NotI restriction site of placz2. ppgkneoa/ls1 was constructed its reading frame (Fig. 1B). Therefore, the F9:LacZ reby cloning the BglII fragment, containing the PGKneo expression porter line should allow the detection of Cre-mediated cassette isolated from pkj-1b [a derivative of pkj1 (22) obtained by converting the EcoRI site into a BglII site] into the BamHI site of recombination by either staining the cells with X-Gal or plox2b. plox2b is a vector containing two direct repeated loxp sites measuring the b-galactosidase activity in cell extracts. flanked by multiple cloning sites and separated by a BamHI site, To evaluate the Cre-mediated induction of b-galacobtained by replacing the sequences located between the NotI and tosidase activity, we established a F9:LacZ:Cre cell line HindIII sites of pbluescript II SK/ (Stratagene) with the following sequence: 5 -TCTAGTATAACTTCGTATAATGTATGCTATACGAAcombinase. This control experiment revealed interestconstitutively expressing the wild-type (WT) Cre re- GT TATCGGATCCGATAACTTCGTATAATGTATGCTATACGAAG- TTATCCCGGGTAGTAGATAGAGATCTGCGGCCGC-3. placz2 was ing properties of the F9:LacZ reporter cell line. As exobtained by introducing a NotI restriction site into the XbaI-HindIII pected F9:LacZ:Cre(WT) cells were neomycin sensitive digested placz1 vector using the oligonucleotides 5 -CTAGAAGGC- (data not shown), in contrast to the parental F9:LacZ GGCCGCTA-3 and 5 -AGCTTAGCGGCCGCCTT-3. placz1 was constructed by ligating the 0.6 kb NsiI-NheI fragment of pbk-cmv cells, indicating that the LoxP-flanked neomycin resis- (Stratagene) containing the cytomegalovirus (CMV) immediate early tance gene was excised. Surprisingly, less than 10% of promoter and the 7.0 kb NsiI-NcoI fragment of pfrtbgal (Stra- F9:LacZ:Cre(WT) cells stained positive for b-galactositagene), treatment with T4 DNA Polymerase to fill in overhanging dase activity, suggesting that the CMV promoter drivbases, followed by a second round of ligation. ing the b-galactosidase gene was not very efficient in The identity of all constructs was confirmed by restriction enzyme mapping and DNA sequence analysis. F9 cells. Interestingly, the expression of b-galactosi- Establishment of stable F9 transfectants. The F9 murine embryonal dase was strongly enhanced by retinoic acid treatment, carcinoma cells were cultured as previously described (14). as more than 70% of the cells became positive when F9 cells were electroporated with 0.5 mg ScaI-linearized grown in the presence of M all-trans retinoic acid placz(lnl) (Fig. 1B). After 24 h, cells were subjected to neomycin (RA) (Fig. 2; that not all cells became positive most selection (660 mg/ml G418 sulfate) for 10 days. Among several neomyprobably reflects some epigenetic variability in the accin-resistant clones that showed b-galactosidase activity after trantivity of the CMV promoter in the F9:LacZ cell line). sient transfection with psgcre, one clone, F9:LacZ, was chosen for a second round of transfection. F9:LacZ cells were coelectroporated Thus, in all further excision experiments, cells were with 0.5 mg PvuII-linearized ppgk-hyg (23) and 2.5 mg of a AseI- grown in the presence of RA. Although measurements linearized plasmid encoding either one of the various mutant Creof b-galactosidase activity do not reflect the absolute ER proteins (Fig. 1A) or AseI-linearized psgcre. Hygromycin seleclevel of recombination in the F9:LacZ reporter cell line, tion (500 mg/ml) was started 24 h after plating and was continued for 10 days. For each cotransfection, three independent hygromycin- this reporter cell line is well suited to compare ligands resistant clones (F9:LacZ:Cre clones) were propagated and assayed for their relative potencies to activate ligand-depenfor b-galactosidase activity in the absence of ligand and in the presdent recombinases. ence of 100 nm E2 or 100 nm OHT. All investigated clones from a given cotransfection showed similar ligand sensitivity and one clone from each cotransfection was selected for further analysis. Cre-ER(GR) Recombinase (Cre-ER T ) Activity Is Detection of b-galactosidase activity. Cells were plated at a den- Inducible by both 4-Hydroxytamoxifen and ICI sity of cells per cm 2 and grown in the presence of 100 nm alltrans retinoic acid (RA). After 24 h cells were exposed to ligand for the 182,780 indicated time period. Cells were analysed 120 hours after plating for Cre-mediated recombination either by direct staining with X-Gal (17) We (15) and others (16) have recently reported Creor by measuring the b-galactosidase activity in cell extracts using ER mutants (Cre-ER T and Cre-TBD, respectively) orthonitrophenyl-b-d-galactoside as a substrate (24). b-galactosi- which are E2-insensitive, but OHT-sensitive. The predase activity was expressed as DOD mg 01 1 h Relative viously described Cre-ER T (15), which consists of Cre b-galactosidase activity was expressed as the percentage of the maxifused to the mutated human ER LBD containing a glymum activity after substraction of the activity obtained in the abcine at amino-acid 400 and an arginine at amino-acid sence of ligand. Each experiment was performed at least in triplicate and mean values ({ 10 %) are given in the figures. 521, is hereafter called Cre-ER(GR) (Fig. 1A). RESULTS We first analysed the recombinase properties of Cre- ER(GR) in the F9:LacZ reporter cell line. Cre-ER(GR) Reporter System for Cre-Mediated Recombination was strongly activated in the presence of the ER antagonists A series of expression vectors were constructed encoding 4-hydroxytamoxifen (OHT) and ICI 182,780 different Cre-ER mutants, each consisting of (ICI), whereas in the absence of exogenous ligands or 753

3 FIG. 1. (A) Structure of Cre-ER constructs. The DNA fragments used for stable expression in F9:LacZ cells contained the simian virus 40 early promoter, the rabbit b-globin intron II, Cre-ER mutants consisting of the Cre recombinase gene fused to cdnas encoding various mutated human ER LBDs, and the polyadenylation site (polya) from the SV40 early region. The nomenclature used for the different Cre- ER proteins and the corresponding amino acid substitutions are shown. Cre-ER (GG) contains the wild-type sequence of the human ER LBD and for each Cre-ER mutant newly introduced amino acids are boxed. Amino acid numbering corresponds to the human ER protein (27). The helixes H10-H12 (28) as well as the regions of the ER LBD that are involved in receptor dimerization (29, 30) and transcriptional transactivation [AF2 AD core; (31, 32)] are indicated. (B) Structure of the LacZ reporter construct before [LacZ(LNL), upper part] and after [LacZ(L); lower part] Cre-mediated excision. The open reading frame of the E. coli b-galactosidase gene (lacz) in the LacZ(LNL) transgene is interrupted by a loxp-flanked PGK promoter-driven neomycin resistance gene (neo). Therefore, RNA transcribed from the cytomegalovirus immediate early promoter (CMV promoter) does not encode functional b-galactosidase. Cre-mediated excision of the neo cassette leaves one loxp site and restores a functional b-galactosidase coding sequence. The amino-terminal b-galactosidase sequence obtained after Cremediated recombination is shown and the amino acids encoded by the loxp sequence are underlined [LacZ(L); lower part]. Also indicated are the positions of the SV40 small t intron and polyadenylation signal (polya), and the RNA startsite (arrow). in the presence of E2, the same low level of b-galactosi- dase activity was detected (Fig. 3 A and B). Further- more, the activation of Cre-ER(GR) by OHT or ICI was not hampered by E2 (Fig. 3B). A time-course experiment (Fig. 3C) revealed that the recombinase activity was rapidly induced by OHT (50% induction after É12 hours) and reached a plateau between 24 and 48 hours. Inducible Recombinase Activity of Novel Cre-ER Mutants The activity of various Cre-ER recombinases was tested in the F9:LacZ line in the absence of exogenous ligand and in the presence of 25 nm E2, OHT or ICI (Fig. 4A). Cre-ER(GG), which contains the wild-type LBD of the human ER showed a high basal activity, comparable to the constitutively active Cre (Fig. 4A). Since the wild-type ER binds E2 with a K D value in the sub-nanomolar range (20, 25), it is likely that Cre- ER(GG) was activated by trace amounts of oestrogens present in the cell culture medium. Similar results were obtained with a FLP-wild-type ER LBD fusion protein (13). The G400V mutation is known to lower the affinity of the human ER for E2 at 37 C (20). In- deed, the corresponding recombinase Cre-ER(VG) gen- erated very little recombination in the absence of added ligand, but was strongly activated by either E2, OHT or ICI (Fig. 4A). Since the activity of Cre-ER(VG) is induced by E2, this recombinase is suitable only for cell culture systems that do not contain E2, but not for mice. To abolish the activation by E2 while retaining the induction by synthetic antagonistic ligands, several mutations were introduced into the Cre-ER recombinase. The G525R mutation of the mouse ER LBD re- duces E2 affinity by appoximately 1000-fold without affecting OHT binding (25). Interestingly, F9:LacZ cells stably transfected with the double mutant Cre-ER(VR) 754

4 that contains the corresponding G521R mutation of the human ER and the G400V substitution (see above), displayed only a low basal recombination activity, and this activity was not increased by any ligand tested (Fig. 4A). Apparently, the ligand affinity was strongly reduced in this double mutant. However, Cre-ER(GR), which contains only the G521R mutation, was insensitive to E2, but was activated by both OHT [as expected from our previous study (15)] and ICI (Fig. 4A, see also Fig. 3). Two other recombinases containing mutations corresponding to mutations reported to abolish activation of the mouse ER by E2, while increasing the potency of OHT to activate the receptor (26) were constructed. Both recombinases, Cre-ER(AA1) (hereafter called Cre-ER T1 ) containing the G400V/L539A/L540A triple mutation of the human ER, and Cre-ER(AA2) (hereafter called Cre-ER T2 ) containing the G400V/ M543A/L540A mutations, were insensitive to E2, but could be efficiently activated by OHT and ICI (Fig. 4A). The ligand inducibilities of Cre-ER T1 and Cre-ER T2 were further investigated and compared to those of Cre-ER(VG) and Cre-ER(GR). To estimate the potency of a given ligand to activate these recombinases, doseresponse experiments were performed (Fig. 4B) and EC 50 values were determined for each ligand/recombinase pair (Table 1). Whereas the E2-sensitive recombinase Cre-ER(VG) was half-maximally activated by É 5 nm OHT, the EC 50 values of the E2-insensitive recombinases Cre-ER(GR) and Cre-ER T1 for OHT were at least 3- to 4-fold higher (Fig. 4B, Table 1). Note that the EC 50 values of the two latter recombinases for OHT most likely represent lower limits, since the corresponding dose-response curves did not clearly reach a plateau level (Fig. 4B). However, Cre-ER T2 was half- FIG. 3. Recombinase activity of Cre-ER(GR) in F9:LacZ:Cre- ER(GR) cells. (A) X-Gal staining of cells cultured for 3 days in the presence of vehicle alone (no ligand), 100 nm E2 or 100 nm OHT. Cells that stained positive for b-galactosidase activity in the absence of ligand or in the presence of E2 are indicated by arrows. (B) b- galactosidase activity in cytosolic extracts of cells treated for 3 days with the indicated concentrations of ligands. (C) Time course of OHTinduced b-galactosidase synthesis. b-galactosidase activity was measured in cytosolic extracts of cells treated for the indicated time periods with 250 nm OHT. For all experiments, F9:LacZ:Cre-ER(GR) cells were cultured in the presence of 100 nm RA. maximally activated at a relatively low concentration of OHT comparable to Cre-ER(VG) (Fig. 4B, Table 1). In contrast to all other E2-insensitive recombinases tested, Cre-ER T1 was highly sensitive to ICI (Table 1). The apparent affinity of Cre-ER T2 for OHT and of Cre- ER T1 for ICI was at least 3 to 4-fold higher than the apparent affinity of Cre-ER(GR) for OHT (Table 1). FIG. 2. Analysis of recombination in the F9:LacZ reporter cell line. X-Gal staining of F9:LacZ:Cre(WT) cells cultured under standard conditions (0RA) or in the presence of 100 nm all-trans retinoic acid (/RA) is presented. DISCUSSION To achieve ligand-dependent site-specific recombina- tion in mice, a Cre-ER recombinase should not be acti- 755

5 vated by endogenous levels of E2 but by low doses of TABLE 1 synthetic ligands like OHT or ICI, to minimize possible Potency of Different Ligands to Activate Cre-ER Mutants side effects. Recently, the E2-insensitive/OHT-inducible recombinases Cre-ER T [called Cre-ER(GR) in this EC 50 (nm) a study] and a similar construct have been described by Cre-ER E2 OHT ICI our group (15) and others (16), respectively. Using the present F9:LacZ reporter line (Fig. 1B), the E2-insensi- (VG) 2 4 c tive/oht-inducible recombination activity of Cre- (GR) b 17 c ER(GR) that was previously detected (15) using other T1 b 20 5 T2 b 6 reporter systems, was confirmed. In addition, we show c here that in F9 cells recombination by Cre-ER(GR) is a Values were determined graphically from dose-response curves also activated by ICI (even in the presence of E2) and performed as described in Fig. 4B. is complete by one day of OHT treatment (Fig. 3). Toligand. c Activity was stimulated, but did not reach a plateau in the pres- b Activity was not stimulated in the presence of up to 250 nm gether, these properties make Cre-ER(GR) a useful tool to achieve conditional recombination in mice. Indeed, ence of up to 250 nm ligand. site-specific recombination has been induced by administration of OHT to transgenic mice expressing Cre- ER(GR) (15). Using the currently available mouse line the level of recombination varies greatly between dif- in some organs (15). This heterogenous recombination ferent tissues and reaches a maximal level of É 50% rate probably reflects variations in CMV-driven Cre- ER(GR) expression. However, the efficiency of liganddependent recombination in an animal might also depend on the potency of the ligand to activate the recombinase. To improve ligand-activated site-specific recombination, a series of chimeric recombinases consisting of Cre fused to mutated human ER LBDs were constructed here and their ligand inducibility characteristics were compared to those of Cre-ER(GR). The recombinase properties of the Cre-ER mutants (Fig. 1A) were analysed by detecting Cre-mediated expression of b-galactosidase activity in the F9:LacZ reporter line (Fig. 1B). For all Cre-ER mutants tested, a low level of b-galactosidase activity in the absence of exogenous ligand was detected. As the parental F9:LacZ reporter line showed no b-galactosidase activity under similar culture conditions (data not shown), this activity represents a low recombination background of the Cre-ER mutants in the absence of their cognate ligands. The basal recombination activity was less than 5% of the activity that was obtained with the fully stimulated ligand-dependent recombinases, and could also be detected by PCR amplification of the recombined lacz transgene (data not shown). Background recombination was also reported in ES cells stably expressing a recombinase similar to Cre-ER(GR) (16) or a fusion protein between Cre and a mutated LBD of the human progesterone receptor (17). Taken together, these results indicate that the expression in FIG. 4. Ligand inducibility of Cre-ER mutants in various cultured cells of the different ligand-dependent Cre re- F9:LacZ:Cre-ER cell lines. (A) Recombinase activity of Cre and varicombinases described to date can lead to recombination ous Cre-ER proteins in the absence of ligand (open bars) and in the presence of 25 nm E2 (filled bar), OHT (hatched bar) or ICI (strippled in the absence of ligand. Interestingly, although recom- bar). (B) Dose-response experiment of the recombinase activity of binase activity in F9:LacZ:Cre-ER(VR) cells was not Cre-ER(VG) ( ), Cre-ER(GR) ( ), Cre-ER T1 ( ), and Cre-ER T2 ( ) stimulated by ligands, a low basal activity similar to in the presence of increasing amounts of OHT. For all experiments that obtained with the ligand-dependent Cre-ER re- F9 cell lines were cultured in the presence of 100 nm RA and b- combinases was detected in these cells (Fig. 4A). As galactosidase activity was measured in cytosolic extracts of cells treated with ligand for 3 days. No b-galactosidase activity was detected in the F9:LacZ cell line. be due to proteolysis of the Cre-LBD fusion suggested by Zhang et al. (16), the basal activity could proteins 756

6 to yield constitutively active products. In transiently 5. Buchholz, F., Ringrose, L., Angrand, P. O., Rossi, F., and Stew- transfected F9 cells and in a transgenic mouse line art, F. (1996) Nucleic Acids Res. 24, Sauer, B., and Henderson, N. (1988) Proc. Natl. Acad. Sci. USA expressing Cre-ER(GR) background recombination 85, was not detected by PCR (15). As the recombinase is 7. Lakso, M., Sauer, B., Mosinger, B., Lee, E. L., Manning, R. W., expressed in only É5% of transiently transfected cells Yu, S. H., Mulder, K. L., and Westphal, H. (1992) Proc. Natl. (unpublished results), the background activity of Cre- Acad. Sci. USA 89, ER(GR) might be below the detection limit. In several 8. Orban, P. C., Chui, D., and Marth, J. D. (1992) Proc. Natl. Acad. tissues of transgenic mice expressing Cre-ER(GR) we Sci. USA 89, observe up to 50% OHT-induced recombination but no 9. Gu, H., Marth, J. D., Orban, P. C., Mossmann, H., and Rajewsky, K. (1994) Science 265, background activity. Thus, the chimeric recombinase 10. Tsien, J. Z., Chen, D. F., Gerber, D., Tom, C., Mercer, E. H., Anmight be less susceptible to proteolysis in mice. derson, D. J., Mayford, M., Kandel, E. R., and Tonegawa, S. The characterization of the Cre-ER mutants described (1996) Cell 87, here indicated that besides Cre-ER(GR), two of the 11. Kühn, R., Schwenk, F., Aguet, M., and Rajewsky, K. (1995) Science newly constructed recombinases (Cre-ER T1 and Cre- 269, ER T2 ) are particularly interesting. These two Cre-ER 12. St-Onge, L., Furth, P. A., and Gruss, P. (1996) Nucleic Acids Res. mutants are indeed activated by the synthetic ligands 24, OHT and ICI, but are insensitive to E2, which is endoge- 13. Logie, C., and Stewart, F. (1995) Proc. Natl. Acad. Sci. USA 92, nously present in mice. Dose-response experiments 14. Metzger, D., Clifford, J., Chiba, H., and Chambon, P. (1995) Proc. showed that Cre-ER T2 and Cre-ER T1 are highly sensitive Natl. Acad. Sci. USA 92, to nanomolar concentrations of OHT and ICI, respec- 15. Feil, R., Brocard, J., Mascrez, B., LeMeur, M., Metzger, D., and tively (Fig. 4). These recombinases are at least 3 to 4- Chambon, P. (1996) Proc. Natl. Acad. Sci. USA 93, fold more sensitive to their cognate ligands compared to Cre-ER(GR) (Table 1). Thus, Cre-ER T2 containing the 16. Zhang, Y., Riesterer, C., Ayrall, A.-M., Sablitzky, F., Littlewood, T. D., and Reth, M. (1996) Nucleic Acids Res. 24, G400V/M543A/L544A triple mutation of the human ER LBD, as well as Cre-ER T1 containing the G400V/L539A/ 17. Kellendonk, C., Tronche, F., Monaghan, A. P., Angrand, P. O., Stewart, F., and Schütz, G. (1996) Nucleic Acids Res. 24, 1404 L540A triple mutation, represent two new ligand-depen dent recombinases that could be very useful for condi- 18. Wakeling, A. E., Dukes, M., and Bowler, J. (1991) Cancer Res. tional somatic mutagenesis in mice. 51, Green, S., Issemann, I., and Scheer, E. (1988) Nucleic Acids Res. 16, 369. ACKNOWLEDGMENTS 20. Tora, L., Mullick, A., Metzger, D., Ponglikitmongkol, M., Park, I., and Chambon, P. (1989) EMBO J. 8, We thank Dr. D. Salin-Drouin and the Laboratoires Besins Is- 21. Kumar, V., Green, S., Staub, A., and Chambon, P. (1986) EMBO covesco for 4-hydroxytamoxifen, A. Wakeling and Zeneca Pharmaceuticals J. 5, for ICI 182,780, P. Unger for excellent technical assistance, 22. McBurney, M. W., Sutherland, L. C., Adra, C. N., Leclair, B., F. Ruffenach for oligonucleotide synthesis, the secretarial staff for Rudnicki, M. A., and Jardine, K. (1991) Nucleic Acids Res. 19, typing, C. Werlé, R. Bucher, B. Boulay and J.M. Lafontaine for pre paring the figures, as well as F.J. Dilworth for critically reading the 23. te Riele, H., Maandag, E. R., Clarke, A., Hooper, M., and Berns, manuscript. Special thanks go to S. Adam for excellent experimental A. (1990) Nature 348, support. This work was supported by funds from the Centre National 24. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular de la Recherche Scientifique, the Institut National de la Santé et de Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory la Recherche Médicale, the Collège de France, the Centre Hospitalier Press, Cold Spring Harbor, NY. Universitaire Régional, the Association pour la Recherche sur le Can- 25. Danielian, P. S., White, R., Hoare, S. A., Fawell, S. E., and cer, the Fondation pour la Recherche Médicale, the Human Frontier Parker, M. G. (1993) Mol. Endocrinol. 7, Science Program and Bristol Myers Squibb. J.W. was supported by 26. Mahfoudi, A., Roulet, E., Dauvois, S., Parker, M. G., and Wahli, fellowships from the Human Capital and Mobility programme and W. (1995) Proc. Natl. Acad. Sci. USA 92, the Fondation pour la Recherche Medicale. R.F. is the recipient of a long-term fellowship from the International Human Frontier Science 27. Green, S., Walter, P., Kumar, V., Krust, A., Bornert, J. M., Argos, Program Organization. P., and Chambon, P. (1986) Nature 320, Wurtz, J. M., Bourget, W., Renaud, J. P., Vivat, V., Chambon, P., Moras, D., and Gronemeyer, H. (1996) Nature Structural Biology REFERENCES 3, Fawell, S. E., Lees, J. A., White, R., and Parker, M. G. (1990) Cell 60, Marth, J. D. (1996) J. Clin. Invest. 97, White, R., Fawell, S. E., and Parker, M. G. (1991) J. Steroid 2. Rajewsky, K., Gu, H., Kühn, R., Betz, U. A. K., Müller, W., Roes, Biochem. Molec. Biol. 40, J., and Schwenk, F. (1996) J. Clin. Invest. 98, Danielian, S. P., White, R., Lees, J. A., and Parker, M. G. (1992) 3. Abremski, K., and Hoess, R. (1984) J. Biol. Chem. 259, 1509 EMBO J. 11, Durand, B., Saunders, M., Gaudon, C., Roy, B., Losson, R., and 4. Cox, M. M. (1983) Proc. Natl. Acad. Sci. USA 80, Chambon, P. (1994) EMBO J. 13,

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