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Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible Cre-ERT and Cre-ERT2 recombinases
Introduction
Materials And Methods
Generation of Cre-ERT and Cre-ERT2 transgenic mice
Preparation and administration of 4-hydroxy-tamoxifen (OHT)
Histochemistry
Results And Discussion
Acknowledgements
References
Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible Cre-ERT and Cre-ERT2 recombinases
Received September 6, 1999; Accepted October 4, 1999
ABSTRACT Conditional DNA excision between two LoxP sites can be achieved in the mouse using Cre-ERT, a fusion protein between a mutated ligand binding domain of the human estrogen receptor (ER) and the Cre recombinase, the activity of which can be induced by 4-hydroxy-tamoxifen (OHT), but not natural ER ligands. We have recently characterized a new ligand-dependent recombinase, Cre-ERT2, which was ~4-fold more efficiently induced by OHT than Cre-ERT in cultured cells. In order to compare the in vivo efficiency of these two ligand-inducible recombinases to generate temporally-controlled somatic mutations, we have engineered transgenic mice expressing a LoxP-flanked (floxed) transgene reporter and either Cre-ERT or Cre-ERT2 under the control of the bovine keratin 5 promoter that is specifically active in the epidermis basal cell layer. No background recombinase activity could be detected, while recombination was induced in basal keratinocytes upon OHT administration. Interestingly, a dose-response study showed that Cre-ERT2 was ~10-fold more sensitive to OHT induction than Cre-ERT.
INTRODUCTION
The efficient introduction of somatic mutations in a given gene, at a given time, in a specific cell type, will greatly facilitate studies of gene function in vertebrates. We have previously established atemporally-controlled site-specific recombination system in mice using a conditional Cre/lox system. The fusion of the Cre recombinase with a mutated ligand binding domain (LBD) of the human estrogen receptor (ER) resulted in a tamoxifen-dependent Cre recombinase, Cre-ERT, that was activated by 4-hydroxy-tamoxifen (OHT), but not by estradiol (1). In mice, the Cre-ERT transgene placed under the control of a cytomegalovirus promoter was expressed in most organs analyzed, but not in all cell types. In the epidermis, Cre-ERT was selectively expressed in the granular cell layer (2). After a few days of tamoxifen treatment, Cre-ERT was translocated from the cytoplasm to the nucleus, and shown to be active in essentially all cells of the granular layer (2), thus indicating that cell-specific expression of Cre-ERT in transgenic mice could be used for the generation of site-specific somatic mutations in a spatio-temporally-controlled manner. Short-term tamoxifen treatments have low acute toxicity and cause no severe abnormalities in mice (3). However, to avoid possible undesired tamoxifen-induced side effects, we recently constructed additional Cre-ER mutants, and showed that, in F9 embryonal carcinoma cells, the OHT sensitivity of the Cre-ERT2 mutant containing the G400V/M543A/L544A triple mutation in the human ER LBD, was ~4-fold higher than that of Cre-ERT (4).
Cells from the basal layer of the skin epidermis undergo a well-defined program of differentiation, coordinated with vertical migration to form suprabasal layers (spinous and granular layers) and a protective cornified layer of enucleated and keratinized cells, which is eventually shed. Thus, epidermal cells are continuously renewed throughout life, utilizing reservoirs of stem cells located in the basal layer and in the outer root sheaths of hair follicles (5). To create temporally-controlled somatic mutations in the epidermis, and to compare the efficiencies of Cre-ERT and Cre-ERT2 in vivo, we have now established transgenic mice expressing these chimeric recombinases under the control of the bovine keratin 5 (K5) promoter which is selectively active in epidermal proliferating basal keratinocytes (6). For each chimeric Cre recombinase, we have selected transgenic lines that exhibit similar patterns and levels of expression. We show that excision of LoxP-flanked (floxed) DNA occurred in cells of the basal layer, upon OHTadministration (1 mg/day for 5 days) to double transgenic mice that carry either one of the two chimeric recombinases together with the Cre recombinase reporter present in the ACZL transgenic line (7). Interestingly, in both cases no background activity could be detected in the absence of OHT administration. However, the OHT dose-response of Cre-ERT2 was ~10-fold more sensitive than that of Cre-ERT for both nuclear translocation and recombinase activity. Thus, Cre-ERT2 may be preferred to generate spatio-temporally-controlled somatic mutations in the mouse, particularly in embryos during the course of gestation. Furthermore, the present K5-Cre-ERT2-expressing transgenic lines provide a tool to generate such mutations in the mouse epidermis.
MATERIALS AND METHODS
Generation of Cre-ERT and Cre-ERT2 transgenic mice
pK5-Cre-ERT and pK5-Cre-ERT2 plasmids were constructed as follows. The 0.85 kb StuI-SalI fragment, containing the rabbit [beta]-globin intron II, a polycloning site and the SV40 polyadenylation signal, isolated from pSG5 (8), was inserted into the StuI-XhoI sites of pMCS-1 (9), resulting in pGS. The 2 kb EcoRI fragments isolated from pCre-ERT (1) and pCre-ERT2 (4) were cloned into the EcoRI site of pGS, resulting in pGS-Cre-ERT and pGS-Cre-ERT2, respectively. The 5.2 kb SalI fragment, containing the bovine keratin K5 promoter and isolated from pK18-BK5P, was then cloned into the SalI site of pGS-Cre-ERT and pGS-Cre-ERT2, yielding pK5-Cre-ERT and pK5-Cre-ERT2, respectively. pK18-BK5P was constructed by cloning the 7 kb KpnI fragment isolated from pCKIII and containing the bovine keratin 5 gene promoter (10,11) into the KpnI site of pK18 (GenBank accession no. SYN8KMRCG). The 8 kb K5-Cre-ERT and K5-Cre-ERT2 fragments were excised from pK5-Cre-ERT and pK5-Cre-ERT2 plasmids by digestion with NotI, purified through a 10-30% sucrose gradient, and injected into C57BL/6× SJL F1 zygotes, as described (1). The Cre-ERT and Cre-ERT2 transgenes were detected in mouse tail DNA by PCR using the primers TK139 (5[prime]-ATTTGCCTGCATTACCGGTC-3[prime]) and TK141 (5[prime]-ATCAACGTTTTCTTTTCGG-3[prime]) located on the 5[prime] and 3[prime] side of the Cre gene, respectively (12). PCR detection of the ACZL transgene and generation of double transgenic mice were as described (2).
Preparation and administration of 4-hydroxy-tamoxifen (OHT)
Ethanol was added to 10 mg of OHT to obtain a 10 mg/100 µl OHT suspension. A 10 mg/ml OHT solution was prepared by addition of autoclaved sunflower oil, followed by 30 min sonication with a Branson ultra-sonicator (Model 1210). Further dilutions were made in autoclaved sunflower oil and stored either at 4°C for a week or at -20°C for months. The OHT stock solutions were sonicated before use, and 100 µl aliquots containing either 1, 0.1 or 0.01 mg OHT were injected intraperitonially into 8-10 week old transgenic mice for 5 consecutive days as described (1).
Histochemistry
Immunohistochemistrywas performed as described (2).For [beta]-galactosidase histochemistry, 10 µm-thick longitudinal sections were cut with a cryostat (Leica CM 3050), stained with X-Gal (5 bromo-4-chloro-3-indolyl [beta]-D-galactoside) as described (13), counterstained with 0.01% safranine, and dehydrated in ethanol (90 and100%; 2 min each) and in LMR-SOL (Labo moderne, 2× 3 min) before mounting the cover-slips.
RESULTS AND DISCUSSION
For both Cre-ERT and Cre-ERT2 transgenes, three transgenic founder animals were identified by PCR analysis of tail DNA. All founders were fertile and yielded transgenic lines. To characterize the expression pattern of the chimeric recombinases, immunohistochemistry analyses were performed on frozen skin sections, using an anti-Cre rabbit polyclonal antibody and confocal microscopy. Using DAPI to stain cell nuclei, basal cells were found to be specifically Cre-positive in the epidermis of all three K5-Cre-ERT and K5-Cre-ERT2 lines (see Fig. 1 and data not shown). Two transgenic lines (one line for each chimeric Cre recombinase), exhibiting similar patterns and levels of Cre expression, were selected for further studies. Both Cre-ERT and Cre-ERT2 proteins were essentially nuclear upon a 5 day treatment with 1 mg OHT/day (Fig. 1), in agreement with our previous results obtained with CMV-Cre-ERT expressing mice in which Cre-ERT was selectively expressed in the granular layer of the epidermis (2). In the absence of ligand treatment both Cre-ERT and Cre-ERT2 proteins were located in the cytoplasm of basal cells, but at a lower level than in the case of transgenic mice expressing Cre-ERT in the granular layer (Fig. 2 and data not shown; see ref. 2).
Figure 1. Expression of Cre-ERT and Cre-ERT2 in tail epidermis of K5-Cre-ERT and K5-Cre-ERT2 transgenic mice. Eight-week-old transgenic mice were injected for 5 consecutive days with OHT (1 mg/day). Immunohistochemistry with an antibody directed against Cre (Cre Ab) was performed on cryosections of tail biopsies of K5-Cre-ERT (a, a[prime]) and K5-Cre-ERT2 (b, b[prime]) transgenic mice, taken 24 h after the last OHT injection. The red color corresponds to the staining of the chimeric recombinases (a, a[prime] and b, b[prime]) and the cyan color to the DAPI staining of the nuclei (a[prime], b[prime]); the white color of the basal cell nuclei (a[prime], b[prime]) results from the superimposition of the red color of the anti-Cre signal and the cyan color of the DAPI staining. B, S and G correspond to the basal, spinous and granular layers, respectively.
Figure 2. Comparison of OHT-induced nuclear translocation of K5-Cre-ERT and K5-Cre-ERT2 proteins. Immunohistochemistry with Cre Ab was performed on cryosections of tail biopsies of 8-week-old K5-Cre-ERT (a-d, a[prime]-d[prime]) and K5-Cre-ERT2 (e-h, e[prime]-h[prime]) double heterozygous transgenic mice. Mice were injected for 2 consecutive days with 1 mg/day (b, b[prime], f, f[prime]), 0.1 mg/day (c, c[prime], g, g[prime]) and 0.01 mg/day (d, d[prime], h, h[prime]) OHT. Tail biopsies were also taken before the first 1 mg OHT injection [(a, a[prime], e, e[prime])]. The cyan color (a-d and e-h) corresponds to the DAPI staining and the red color (a-d, a[prime]-d[prime] and e-h, e[prime]-h[prime]) corresponds to the staining of the Cre-ERT and Cre-ERT2, respectively.
To estimate the binding of OHT to Cre-ERT and Cre-ERT2, we compared the intracellular localization of the chimeric proteins upon treatment of the transgenic mice with decreasing doses of OHT. Two days of 1 mg OHT treatment were sufficient to induce an efficient nuclear translocation of both Cre-ERT and Cre-ERT2 proteins (Fig. 2, compare panels a and a[prime] and b and b[prime], with panels e and e[prime] and f and f[prime], respectively). In contrast, a 2 day 0.1 mg OHT treatment resulted in a stronger nuclear localization for Cre-ERT2 than for Cre-ERT that was fully translocated into nuclei after 5 days of treatment only (Fig. 2, compare panels g and g[prime] and panels c and c[prime], respectively; and data not shown). A 2 day 0.01 mg OHT treatment was sufficient to translocate some Cre-ERT2 into nuclei (panels h and h[prime]), while no Cre-ERT positive nuclei could be observed (panels d and d[prime]), even upon a 5 day treatment (data not shown), clearly indicating that lower doses of OHT were required to translocate Cre-ERT2 into basal cell nuclei.
To compare the recombinase activity of the two chimeric recombinases, K5-Cre-ERT and K5-Cre-ERT2 transgenic mice were crossed with the ACZL Cre recombinase reporter mouse line (7). In this line the expression of a loxP-chloramphenicol acetyl transferase (CAT) cassette-loxP-lacZ cassette is driven by the CMV enhancer and the chicken [beta]-actin promoter (14), and [beta]-galactosidase is produced only after Cre-mediated excision of the loxP-flanked (floxed) CAT cassette. However, due to cell-type restricted promoter activity (7 and data not shown), the Cre recombinase reporter transgene is not expressed in all cell types, and within the epidermis it is selectively expressed in keratinocytes of the granular layer (2 and data not shown). K5-Cre-ERT/ACZL and K5-Cre-ERT2/ACZL double heterozygote transgenic mice were examined for Cre-mediated excision upon 1, 0.1 and 0.01 mg OHT treatments. To this end, mice were injected for 5 days with OHT, and X-Gal staining was performed on tail skin sections 10 days after the last injection, in order to allow basal cells to differentiate into granular cells. Whereas no X-Gal staining could be detected in the skin of untreated or vehicle-treated K5-Cre-ERT/ACZL and K5-Cre-ERT2/ACZL mice, 1 mg OHT treatment resulted in both cases in blue staining of suprabasal cells (Fig. 3 and data not shown). Similar blue stainings were observed 25 days after the last OHT injection (data not shown). As suprabasal cells are renewed in 7-10 days in the mouse tail epidermis (2 and data not shown), these results show that recombination had occurred in proliferative basal cells, whereas the lack of blue staining in the absence of ligand treatment clearly indicates that the recombinase activities of both Cre-ERT and Cre-ERT2 are fully dependent on OHT binding. Interestingly, the X-Gal staining pattern of the epidermis of K5-Cre-ERT2 mice was very similar upon a 0.1 mg OHT treatment, while upon the same treatment not more than 30% of the suprabasal cells of K5-Cre-ERT mouse epidermis was stained (Fig. 3 and data not shown). When 0.01 mg OHT was used, no X-Gal staining could be observed in K5-Cre-ERT epidermis, whereas in the case of K5-Cre-ERT2 mice the staining was similar to that seen in 0.1 mg OHT-treated K5-Cre-ERT mice (Fig. 3 and data not shown).
Figure 3. Comparison of the OHT-induced expression of [beta]-galactosidase in tail epidermis of K5-Cre-ERT/ACZL (a-d) and K5-Cre-ERT2/ACZL (a[prime]-d[prime]) double heterozygous transgenic mice. Eight-week-old K5-Cre-ERT/ACZL (a-d) and K5-Cre-ERT2/ACZL (a[prime]-d[prime]) double heterozygous mice were injected daily with 1, 0.1 and 0.01 mg/day OHT from day 0 to 4. Tail biopsies collected just before the first OHT injection (0; a, a[prime]) or at day 15 following 1 mg (b, b[prime]), 0.1 mg (c, c[prime]) and 0.01 mg (d, d[prime]) OHT injection, were stained with X-Gal (Materials and Methods). Arrows point to the basement membrane. B and S correspond to the basal and suprabasal layers, respectively.
Taken all together, our results show that the recombinase activity of Cre-ERT2 can be induced in the mouse with OHT doses that are ~10-fold lower than those required to activate Cre-ERT. Upon oral or topical tamoxifen administration, Vasioukhin et al. (15) recently reported an efficient excision of loxP-flanked sequences in epidermis basal cells of adult mice expressing another Cre-ER fusion protein, Cre-ERtam, under the control of the human K14 keratin promoter. CreERtam is similar to Cre-ERT, but contains the mouse, instead of the human, mutated ER LBD. Danielian et al. (16) have reported that the dose of ligand required to fully activate Cre-ERTM (another Cre-ERT-like tamoxifen-inducible Cre recombinase) in developing mouse embryos is close to that which interferes with the maintenance of pregnancy. Thus, the present conditional Cre-ERT2 recombinase should be preferentially used to generate spatio-temporally controlled somatic mutations in the mouse, whenever tamoxifen administration may result in undesirable side effects, notably during gestation.
ACKNOWLEDGEMENTS
We are grateful to M. Blessing for the bovine K5 promoter and to A. Berns and M. Giovannini for ACZL mice. We thank S. Bronner, C. Gérard and N. Messaddeq for excellent technical help, M. LeMeur and the animal facility staff for animal care, the secretarial staff for typing the manuscript and the illustration staff for preparing the figures. This work was supported by funds from the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, the Collège de France, the Hôpital Universitaire de Strasbourg, the Association pour la Recherche sur le Cancer, the Fondation pour la Recherche Médicale, the Human Frontier Science Program, the European Economic Community contract FAIR-CT97-3220 and the Ministère de l'Éducation Nationale de la Recherche et de la Technologie Déc.97.C.0115. A.K.I. was supported by a fellowship from the Université Louis Pasteur (Strasbourg), and J.B. and X.W. by fellowships from the Ministère de l'Education Nationale, de la Recherche et de la Technologie and from the Fondation pour la Recherche Médicale.
REFERENCES
*To whom correspondence should be addressed. Tel: +33 3 88 65 32 13; Fax: +33 3 88 65 32 03; Email: chambon{at}igbmc.u-strasbg.fr
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T. Oskarsson, M. A. G. Essers, N. Dubois, S. Offner, C. Dubey, C. Roger, D. Metzger, P. Chambon, E. Hummler, P. Beard, et al. Skin epidermis lacking the c-myc gene is resistant to Ras-driven tumorigenesis but can reacquire sensitivity upon additional loss of the p21Cip1 gene Genes & Dev., August 1, 2006; 20(15): 2024 - 2029. [Abstract] [Full Text] [PDF]
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 I. J. Huijbers, P. Krimpenfort, P. Chomez, M. A. van der Valk, J.-Y. Song, E.-M. Inderberg-Suso, A.-M. Schmitt-Verhulst, A. Berns, and B. J. Van den Eynde An Inducible Mouse Model of Melanoma Expressing a Defined Tumor Antigen. Cancer Res., March 15, 2006; 66(6): 3278 - 3286. [Abstract] [Full Text] [PDF]
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T. Prathapam, S. Tegen, T. Oskarsson, A. Trumpp, and G. S. Martin Activated Src abrogates the Myc requirement for the G0/G1 transition but not for the G1/S transition PNAS, February 21, 2006; 103(8): 2695 - 2700. [Abstract] [Full Text] [PDF]
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S. Berger, D. P. Wolfer, O. Selbach, H. Alter, G. Erdmann, H. M. Reichardt, A. N. Chepkova, H. Welzl, H. L. Haas, H.-P. Lipp, et al. Loss of the limbic mineralocorticoid receptor impairs behavioral plasticity PNAS, January 3, 2006; 103(1): 195 - 200. [Abstract] [Full Text] [PDF]
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A. K. Indra, V. Dupe, J.-M. Bornert, N. Messaddeq, M. Yaniv, M. Mark, P. Chambon, and D. Metzger Temporally controlled targeted somatic mutagenesis in embryonic surface ectoderm and fetal epidermal keratinocytes unveils two distinct developmental functions of BRG1 in limb morphogenesis and skin barrier formation Development, October 15, 2005; 132(20): 4533 - 4544. [Abstract] [Full Text] [PDF]
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 A. Olry, P. Chastagner, A. Israel, and C. Brou Generation and Characterization of Mutant Cell Lines Defective in {gamma}-Secretase Processing of Notch and Amyloid Precursor Protein J. Biol. Chem., August 5, 2005; 280(31): 28564 - 28571. [Abstract] [Full Text] [PDF]
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M. M. Riccomagno, S. Takada, and D. J. Epstein Wnt-dependent regulation of inner ear morphogenesis is balanced by the opposing and supporting roles of Shh Genes & Dev., July 1, 2005; 19(13): 1612 - 1623. [Abstract] [Full Text] [PDF]
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B. Vernay, M. Koch, F. Vaccarino, J. Briscoe, A. Simeone, R. Kageyama, and S.-L. Ang Otx2 Regulates Subtype Specification and Neurogenesis in the Midbrain J. Neurosci., May 11, 2005; 25(19): 4856 - 4867. [Abstract] [Full Text] [PDF]
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G. W. McLean, N. H. Komiyama, B. Serrels, H. Asano, L. Reynolds, F. Conti, K. Hodivala-Dilke, D. Metzger, P. Chambon, S. G.N. Grant, et al. Specific deletion of focal adhesion kinase suppresses tumor formation and blocks malignant progression Genes & Dev., December 15, 2004; 18(24): 2998 - 3003. [Abstract] [Full Text] [PDF]
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 J.-E. Kim, K. Nakashima, and B. de Crombrugghe Transgenic Mice Expressing a Ligand-Inducible Cre Recombinase in Osteoblasts and Odontoblasts: A New Tool to Examine Physiology and Disease of Postnatal Bone and Tooth Am. J. Pathol., December 1, 2004; 165(6): 1875 - 1882. [Abstract] [Full Text] [PDF]
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 A. Gawlik and S. E. Quaggin Deciphering the Renal Code: Advances in Conditional Gene Targeting Physiology, October 1, 2004; 19(5): 245 - 252. [Abstract] [Full Text] [PDF]
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K. G. Wirth, R. Ricci, J. F. Gimenez-Abian, S. Taghybeeglu, N. R. Kudo, W. Jochum, M. Vasseur-Cognet, and K. Nasmyth Loss of the anaphase-promoting complex in quiescent cells causes unscheduled hepatocyte proliferation Genes & Dev., January 1, 2004; 18(1): 88 - 98. [Abstract] [Full Text] [PDF]
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 A. Ouvrard-Pascaud, S. Puttini, Y. Sainte-Marie, R. Athman, V. Fontaine, F. Cluzeaud, N. Farman, M.-E. Rafestin-Oblin, M. Blot-Chabaud, and F. Jaisser Conditional gene expression in renal collecting duct epithelial cells: use of the inducible Cre-lox system Am J Physiol Renal Physiol, January 1, 2004; 286(1): F180 - F187. [Abstract] [Full Text] [PDF]
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M. Allen, M. Grachtchouk, H. Sheng, V. Grachtchouk, A. Wang, L. Wei, J. Liu, A. Ramirez, D. Metzger, P. Chambon, et al. Hedgehog Signaling Regulates Sebaceous Gland Development Am. J. Pathol., December 1, 2003; 163(6): 2173 - 2178. [Abstract] [Full Text]
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 S. WERNER and R. GROSE Regulation of Wound Healing by Growth Factors and Cytokines Physiol Rev, July 1, 2003; 83(3): 835 - 870. [Abstract] [Full Text] [PDF]
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 J. Seibler, B. Zevnik, B. Kuter-Luks, S. Andreas, H. Kern, T. Hennek, A. Rode, C. Heimann, N. Faust, G. Kauselmann, et al. Rapid generation of inducible mouse mutants Nucleic Acids Res., February 15, 2003; 31(4): e12 - e12. [Abstract] [Full Text] [PDF]
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 P. Weber, M. Schuler, C. Gerard, M. Mark, D. Metzger, and P. Chambon Temporally Controlled Site-Specific Mutagenesis in the Germ Cell Lineage of the Mouse Testis Biol Reprod, February 1, 2003; 68(2): 553 - 559. [Abstract] [Full Text] [PDF]
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E. Bockamp, M. Maringer, C. Spangenberg, S. Fees, S. Fraser, L. Eshkind, F. Oesch, and B. Zabel Of mice and models: improved animal models for biomedical research Physiol Genomics, December 3, 2002; 11(3): 115 - 132. [Abstract] [Full Text] [PDF]
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L. van der Weyden, D. J. Adams, and A. Bradley Tools for targeted manipulation of the mouse genome Physiol Genomics, December 3, 2002; 11(3): 133 - 164. [Abstract] [Full Text] [PDF]
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M. Mori, D. Metzger, J.-M. Garnier, P. Chambon, and M. Mark Site-Specific Somatic Mutagenesis in the Retinal Pigment Epithelium Invest. Ophthalmol. Vis. Sci., May 1, 2002; 43(5): 1384 - 1388. [Abstract] [Full Text] [PDF]
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B. Zheng, Z. Zhang, C. M. Black, B. de Crombrugghe, and C. P. Denton Ligand-Dependent Genetic Recombination in Fibroblasts : A Potentially Powerful Technique for Investigating Gene Function in Fibrosis Am. J. Pathol., May 1, 2002; 160(5): 1609 - 1617. [Abstract] [Full Text] [PDF]
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F. T. Wunderlich, H. Wildner, K. Rajewsky, and F. Edenhofer New variants of inducible Cre recombinase: a novel mutant of Cre-PR fusion protein exhibits enhanced sensitivity and an expanded range of inducibility Nucleic Acids Res., May 15, 2001; 29(10): e47 - e47. [Abstract] [Full Text] [PDF]
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 T. Minamino, V. Gaussin, F. J. DeMayo, and M. D. Schneider Inducible Gene Targeting in Postnatal Myocardium by Cardiac-Specific Expression of a Hormone-Activated Cre Fusion Protein Circ. Res., March 30, 2001; 88(6): 587 - 592. [Abstract] [Full Text] [PDF]
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M Li, H Chiba, X Warot, N Messaddeq, C Gerard, P Chambon, and D Metzger RXR-alpha ablation in skin keratinocytes results in alopecia and epidermal alterations Development, January 3, 2001; 128(5): 675 - 688. [Abstract] [PDF]
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T. Imai, M. Jiang, P. Chambon, and D. Metzger Impaired adipogenesis and lipolysis in the mouse upon selective ablation of the retinoid X receptor alpha mediated by a tamoxifen-inducible chimeric Cre recombinase (Cre-ERT2) in adipocytes PNAS, December 22, 2000; (2000) 11528898. [Abstract] [Full Text]
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E. Fuhrmann-Benzakein, I. Garcia-Gabay, M. S. Pepper, J.-D. Vassalli, and P. L. Herrera Inducible and irreversible control of gene expression using a single transgene Nucleic Acids Res., December 1, 2000; 28(23): e99 - e99. [Abstract] [Full Text] [PDF]
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 F. JAISSER Inducible Gene Expression and Gene Modification in Transgenic Mice J. Am. Soc. Nephrol., November 1, 2000; 11(90002): S95 - S100. [Abstract] [Full Text] [PDF]
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T. Imai, M. Jiang, P. Chambon, and D. Metzger Impaired adipogenesis and lipolysis in the mouse upon selective ablation of the retinoid X receptor alpha mediated by a tamoxifen-inducible chimeric Cre recombinase (Cre-ERT2) in adipocytes PNAS, January 2, 2001; 98(1): 224 - 228. [Abstract] [Full Text] [PDF]
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