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).

Nuclear receptors for retinoids (RARs) and vitamin D (VDR), and for some other ligands (TRs, PPARs and LXRs), maybe critical in the development and homeostasis of mammalian epidermis. It is believed that these receptors form heterodimers with retinoid X receptors (RXRs) to act as transcriptional regulators. However, most genetic approaches aimed at establishing their physiological functions in the skin have been inconclusive owing either to pleiotropic effects and redundancies between receptor isotypes in gene knockouts, or to equivocal interpretation of dominant-negative mutant studies in transgenic mice. Moreover, knockout of RXRalpha, the main skin RXR isotype, is lethal in utero before skin formation. Here we have resolved these problems by developing an efficient technique to create spatiotemporally controlled somatic mutations in the mouse. We used tamoxifen-inducible Cre-ER(T) recombinases to ablate RXRalpha selectively in adult mouse keratinocytes. We show that RXRalpha has key roles in hair cycling, probably through RXR/VDR heterodimers, and in epidermal keratinocyte proliferation and differentiation.

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-ER T ), which binds tamoxifen but not estrogens. DNA excision was induced in a number of tissues after administration of tamoxifen to transgenic mice expressing Cre-ER T under the control of the cytomegalovirus promoter. However, the efficiency of excision varied between tissues, and the highest level (Ϸ40%) was obtained in the skin. To determine the efficiency of excision mediated by Cre-ER T in a given cell type, we have now crossed Cre-ER T -expressing mice with reporter mice in which expression of Escherichia coli ␤-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-ER T . These results indicate that cell-specific expression of Cre-ER T in transgenic mice can be used for efficient tamoxifendependent, Cre-mediated recombination at loci containing loxP sites to generate site-specific somatic mutations in a spatio-temporally controlled manner.