Consecutive inactivation of both alleles of the gb110 gene has no effect on the proliferation and differentiation of mouse embryonic stem cells

Abstract

The gene gb110 was originally identified by provirus integration in the Mov10 mouse strain and encodes a 110-kDa protein with potential GTP-binding activity. The gene is evolutionarily conserved, and its expression is controlled in a developmentally and cell-cycle-specific manner, suggesting that it has an important function in differentiation and development. As a first step in studying the functional role of gb110, embryonal stem (ES) cell lines were derived in which both gb110 alleles were inactivated by consecutive gene targeting via homologous recombination. The first allele was interrupted by integration of a neomycin resistance-encoding gene ( neo) and, subsequently, the second allele by integration of a hygromycin B resistance-encoding gene ( hyg). Selection for homologous recombination was achieved by using promoter and AUG codon-deficient hyg or neo whose expression was dependent on integration into the host genome next to the transcriptional and translational start signals. The efficiency of gb110 gene targeting was very high, with 85–100% of all drug-resistant colonies having undergone homologous recombination. ES cells lacking a functional gb110 were indistinguishable from the wild-type ES cells, indicating that this gene is not required for normal ES cell proliferation and differentiation in vitro.