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      Inducible Cre/loxP Recombination in the Mouse Proximal Tubule

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          Transgenic technologies in mice became invaluable experimental tools to identify the in vivo function of proteins. However, conventional knockout technology often results in embryonic lethality and because genes are frequently expressed in multiple cell types, the resulting knockout phenotypes can be complex and difficult or impossible to dissect. These issues are particularly important for gene-targeting strategies used to examine renal function. The kidney contains quite a number of different cell types, the function of many of which impacts that of other renal cells. To avoid these limitations conditional knockout strategies have been designed. As one important part of this system we describe the development of a mouse line expressing the tamoxifen-activatable Cre recombinase Cre-ER<sup>T2</sup> specifically in renal proximal tubules. The expression of Cre-ER<sup>T2</sup> is driven by a promoter fragment of the mouse γ-glutamyl transpeptidase type II gene resulting in the generation of the activatable recombinase in S3 segments of the proximal tubules from which over 80% were positive for Cre activity. In combination with loxP-based conditional mutant mice as a second tool this tamoxifen-inducible Cre-ER<sup>T2</sup> line allows functional analysis of a variety of genes important for renal development and function in a precisely controlled spatiotemporal manner.

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          Most cited references 26

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          Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains.

          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 mutated 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 17 beta-oestradiol (E2). Both chimeric recombinases should be useful for efficient spatio-temporally controlled site-directed somatic mutagenesis.
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            Tight control of gene expression in mammalian cells by tetracycline-responsive promoters.

             M Gossen,  H Bujard (1992)
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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-ER(T) and Cre-ER(T2) recombinases.

              Conditional DNA excision between two LoxP sites can be achieved in the mouse using Cre-ER(T), 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-ER(T2), which was approximately 4-fold more efficiently induced by OHT than Cre-ER(T) 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-ER(T) or Cre-ER(T2) 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-ER(T2) was approximately 10-fold more sensitive to OHT induction than Cre-ER(T).

                Author and article information

                Nephron Exp Nephrol
                Cardiorenal Medicine
                S. Karger AG
                May 2007
                12 March 2007
                : 106
                : 1
                : e11-e20
                aInstitut für Humangenetik, Universitätsklinikum Münster/Westfälische Wilhelms-Universität und bInstitut für Experimentelle Pathologie (ZMBE), Universitätsklinikum Münster, Münster, Deutschland; cInstitut Clinique de la Souris et dInstitut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France
                100554 Nephron Exp Nephrol 2007;106:e11–e20
                © 2007 S. Karger AG, Basel

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                Page count
                Figures: 4, References: 40, Pages: 1
                Original Paper

                Cardiovascular Medicine, Nephrology

                Tamoxifen, Kidney, Cre-ERT2 , Cre recombinase


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