DISRUPTION OF GENES REGULATED DURING HEMATOPOIETIC DIFFERENTIATION OFMOUSE EMBRYONIC STEM-CELLS

A retroviral gene trap vector (U3Tkneo) that selects for integrations in or near expressed 5' exons has been used to identify genes that are repressed during hematopoietic differentiation of mouse totipotent em bryonic stem cells. The vector contains coding sequences for an HSV-th ymidine kinase/neomycin phosphotransferase fusion protein in the U3 re gion of a Moloney murine leukemia virus LTR and allows selection for ( G418) and against (Ganciclovir; GC) U3 gene expression. A total of 208 neomycin-resistant clones were isolated following infection with U3tk neo and screened for integrations into regulated genes by using a two- step, semisolid culture system that supports hematopoietic differentia tion. Two clones contained U3Tkneo integrations in genes that were rep ressed selectively in hematopoietic cells. Analysis of upstream provir al flanking sequences indicated that both integrations occurred into u nknown genes, One upstream sequence identified a cellular transcript t hat was expressed differentially in the kidneys and liver of adult mic e. When this fusion gene was passaged to the germ line, homozygous off spring with nearly null mutations were obtained. However, mutant mice were normal, suggesting that potential loss of function phenotypes are subtle and may be restricted to the kidneys and the liver. (C) 1998 W iley-Liss, Inc.