Disruption of the gene encoding the ubiquitin-conjugating enzyme UbcM4 has no effect on proliferation and in vitro differentiation of mouse embryonic stem cells.
The ubiquitin-conjugating enzyme UbcM4, which is identical to the human enzyme UbcH7, was previously shown to be essential for normal mouse development. In order to study the possible role of UbcM4 for cell proliferation and in vitro differentiation, we here describe the establishment and characterization 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 growth rates, cell cycle progression and in vitro differentiation when compared 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 mutation. As in the mutant embryos, steady levels of RNA and protein in the cell lines were reduced by about 70%. The mutant cell lines showed no differences in immortalization kinetics, growth rates and cell cycle progression when compared to wild-type fibroblasts. Taken together, our results strongly suggest that UbcM4-mediated ubiquitination and degradation are not necessary for proteins involved in the maintenance and growth of cells.
Pringa
E
E
Heinrich-Pette-Institut für experimentelle Virologie und Immunologie an der Universität Hamburg, Martinistr. 52, D-20251, Hamburg, Germany.
Meier
I
I
Müller
U
U
Martinez-Noel
G
G
Harbers
K
K
D000818:Animals; D015153:Blotting, Western; D002453:Cell Cycle; D002454:Cell Differentiation; D002455:Cell Division; D002478:Cells, Cultured; D005434:Flow Cytometry; D017353:Gene Deletion; D005838:Genotype; D008025:Ligases / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008815:Mice, Inbred Strains; D010641:Phenotype; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes; D014452:Ubiquitins / Q000378:metabolism
D012333:RNA, Messenger; D014452:Ubiquitins; C117188:Ube2l3 protein, mouse; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases