Homologous recombination efficiency enhanced by inhibition of MEK and GSK3β.

Abstract

Homologous recombination in embryonic stem cells (ESCs) is widely utilized in genome engineering, particularly in the generation of gene targeted mice. However, genome engineering is often plagued by the problem of low homologous recombination efficiency. In this study, we developed a novel method to increase the efficiency of homologous recombination in ESCs by changing its culture conditions. By comparing the efficiency of different ESCs in various culture conditions, we determined that chemicals that inhibit the MEK and GSK3β pathways (2i condition) enhance homologous recombination and eliminate differences in efficiencies among cell lines. Analysis of gene expression patterns in ESCs maintained in different culture conditions has identified several homologous recombination-related candidates, including the pluripotent markers Eras and Tbx3. The results of this study suggest that homologous recombination is associated with ESC pluripotency.