Spatio-temporally controlled site-specific somatic mutagenesis 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 shown previously that conditional recombination-excision between two loxP sites can be achieved in mice by using the Cre recombinase fused to a mutated ligand binding domain of the human estrogen receptor (Cre-ERT), which binds tamoxifen but not estrogens. DNA excision was induced in a number of tissues after administration of tamoxifen to transgenic mice expressing Cre-ERT under the control of the cytomegalovirus promoter. 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-ERT in a given cell type, we have now crossed Cre-ERT-expressing mice 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. We show that site-specific excision occurred within a few days of tamoxifen treatment in essentially all epidermis cells expressing Cre-ERT. These results indicate that cell-specific expression of Cre-ERT 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 spatio-temporally controlled manner.
Brocard
J
J
Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Université Louis Pasteur, Collège de France, Strasbourg, France.
Warot
X
X
Wendling
O
O
Messaddeq
N
N
Vonesch
J L
JL
Chambon
P
P
Metzger
D
D
D000818:Animals; D004817:Epidermis / Q000378:metabolism; D018390:Gene Targeting; D006801:Humans; D019426:Integrases / Q000235:genetics*; D051379:Mice; D008822:Mice, Transgenic; D016297:Mutagenesis, Site-Directed; D011960:Receptors, Estrogen / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D013629:Tamoxifen / Q000378:metabolism; D014764:Viral Proteins
D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D013629:Tamoxifen; C045073:Cre recombinase; D019426:Integrases