Site- and time-specific gene targeting in the mouse.
Fiche publication
Date publication
mai 2001
Journal
Methods (San Diego, Calif.)
Auteurs
Membres identifiés du Cancéropôle Est :
Pr CHAMBON Pierre, Dr METZGER Daniel
Tous les auteurs :
Metzger D, Chambon P
Lien Pubmed
Résumé
The efficient introduction of somatic mutations in a given gene, at a given time, in a specific cell type, will facilitate studies of gene function and the generation of animal models for human diseases. We have established a conditional site-specific recombination system in mice using a new version of the Cre/lox system. The Cre recombinase has been fused to a mutated ligand binding domain of the human estrogen receptor (ER), resulting in a tamoxifen-dependent Cre recombinase, Cre-ER(T), that is activated by tamoxifen, but not by estradiol. Transgenic mice were generated expressing Cre-ER(T) under the control of a cytomegalovirus promoter. Administration of tamoxifen to these transgenic mice induced excision of a chromosomally integrated gene flanked by loxP sites in a number of tissues, whereas no excision could be detected in untreated animals. However, the efficiency of excision varied between tissues, and the highest level (approximately 40%) was obtained in the skin. To determine the efficiency of excision mediated by Cre-ER(T) in a given cell type, Cre-ER(T)-expressing mice were crossed with reporter mice in which expression of Escherichia coli beta-galactosidase can be induced through Cre-mediated recombination. The efficiency and kinetics of this recombination were analyzed at the cellular level in the epidermis of 6- to 8-week-old double transgenic mice. Site-specific excision occurred within a few days of tamoxifen treatment in essentially all epidermis cells expressing Cre-ER(T). These results indicate that cell-specific expression of Cre-ER(T) in transgenic mice can be used for efficient tamoxifen-dependent Cre-mediated recombination at loci containing loxP sites, to generate site-specific somatic mutations in a spatiotemporally controlled manner. This conditional site-specific recombination system should allow the analysis of knockout phenotypes that cannot be addressed by conventional gene targeting.
Mots clés
Alleles, Animals, Antineoplastic Agents, Hormonal, pharmacology, Cell Nucleus, metabolism, Crosses, Genetic, Cytoplasm, metabolism, Epidermis, metabolism, Escherichia coli, enzymology, Gene Transfer Techniques, Humans, Integrases, metabolism, Ligands, Mice, Mice, Knockout, Mice, Transgenic, Models, Genetic, Promoter Regions, Genetic, Receptors, Estrogen, genetics, Recombination, Genetic, Tamoxifen, pharmacology, Time Factors, Viral Proteins, beta-Galactosidase, metabolism
Référence
Methods. 2001 May;24(1):71-80