Disruption of the gene encoding the ubiquitin-conjugating enzyme UbcM4 hasno effect on proliferation and in vitro differentiation of mouse embryonicstem cells

The ubiquitin-conjugating enzyme UbcM4, which is identical to the human enz yme UbcH7, was previously shown to be essential for normal mouse developmen t. In order to study the possible role of UbcM4 for cell proliferation and in vitro differentiation, we here describe the establishment and characteri zation of fibroblast and embryonic stem cell lines with partial or complete inactivation of the UbcM4 gene. ES cell lines in which both alleles of the gene were inactivated by targeted mutagenesis showed no differences in gro wth rates, cell cycle progression and in vitro differentiation when compare d to wild-type ES cells. Fibroblast cell lines with a partially inactivated UbcM4 gene were derived from embryos of the previously described A6 mouse mutant, where retrovirus integration has resulted in a recessive lethal mut ation. As in the mutant embryos, steady levels of RNA and protein in the ce ll lines were reduced by about 70%. The mutant cell lines showed no differe nces in immortalization kinetics, growth rates and cell cycle progression w hen compared to wild-type fibroblasts. Taken together, our results strongly suggest that UbcM4-mediated ubiquitination and degradation are not necessa ry for proteins involved in the maintenance and growth of cells. (C) 2000 E lsevier Science B.V. All rights reserved.