Site- and time-specific gene targeting in the mouse.
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.
Metzger
D
D
Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, BP 163 67404 Illkirch Cedex, C.U. de Strasbourg, France.
Chambon
P
P
D000483:Alleles; D000818:Animals; D018931:Antineoplastic Agents, Hormonal / Q000494:pharmacology; D002467:Cell Nucleus / Q000378:metabolism; D003433:Crosses, Genetic; D003593:Cytoplasm / Q000378:metabolism; D004817:Epidermis / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D018014:Gene Transfer Techniques; D006801:Humans; D019426:Integrases / Q000378:metabolism; D008024:Ligands; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D008957:Models, Genetic; D011401:Promoter Regions, Genetic; D011960:Receptors, Estrogen / Q000235:genetics; D011995:Recombination, Genetic; D013629:Tamoxifen / Q000494:pharmacology; D013997:Time Factors; D014764:Viral Proteins; D001616:beta-Galactosidase / Q000378:metabolism
D018931:Antineoplastic Agents, Hormonal; D008024:Ligands; D011960:Receptors, Estrogen; D014764:Viral Proteins; D013629:Tamoxifen; C045073:Cre recombinase; D019426:Integrases; D001616:beta-Galactosidase