An efficient system for conditional gene expression in embryonic stem cells and in their in vitro and in vivo differentiated derivatives

We have developed a universally applicable system for conditional gene expr ession in embryonic stem (ES) cells that relies on tamoxifen-dependent Cre recombinase-loxP site-mediated recombination and bicistronic gene-trap expr ession vectors that allow transgene expression from endogenous cellular pro moters. Two vectors were introduced into the genome of recipient ES cells, successively: (i) a bicistronic gene-trap vector encoding the beta -galacto sidase/ neo(R) fusion protein and the Cre-ERT2 (Cre recombinase fused to a mutated ligand-binding domain of the human estrogen receptor) and (ii) a bi cistronic gene-trap vector encoding the hygro(R) protein and the human alka line phosphatase (hAP), the expression of which is prevented by tandemly re peated stop-of-transcription sequences flanked by loxP sites. In selected c lones, hAP expression was shown to be regulated accurately by 4'hydroxy-tam oxifen. Strict hormone-dependent expression of hAP was achieved (i) in vitr o in undifferentiated ES cells and embryoid bodies, (ii) in vivo in virtual ly all the tissues of the 10-day-old chimeric fetus (after injection of 4'h ydroxy-tamoxifen to foster mothers), and (iii) ex vivo in primary embryonic fibroblasts isolated from chimeric fetuses. Therefore, this approach can b e applied to drive conditional expression of virtually any transgene in a l arge variety of cell types, both in vitro and in vivo.