title: 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.
authors.affiliation: MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center of Nanjing University, Nanjing, Jiangsu, 210061, People's Republic of China.
authors.forename: Zhaoyu
authors.initials: Z
authors.lastname: Lin
authors.forename: Yanli
authors.initials: Y
authors.lastname: Zhang
authors.forename: Tianyun
authors.initials: T
authors.lastname: Gao
authors.forename: Liudi
authors.initials: L
authors.lastname: Wang
authors.forename: Qing
authors.initials: Q
authors.lastname: Zhang
authors.forename: Juan
authors.initials: J
authors.lastname: Zhou
authors.forename: Jing
authors.initials: J
authors.lastname: Zhao
mesh_terms: D000818:Animals; D001549:Benzamides / Q000494:pharmacology; D015153:Blotting, Western; D018929:Cell Culture Techniques / Q000379:methods*; D004159:Diphenylamine / Q000031:analogs & derivatives / Q000494:pharmacology; D053595:Embryonic Stem Cells / Q000502:physiology*; D018390:Gene Targeting / Q000379:methods; D005818:Genetic Engineering / Q000379:methods*; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors*; D000071679:Glycogen Synthase Kinase 3 beta; D059765:Homologous Recombination / Q000235:genetics / Q000502:physiology*; D007150:Immunohistochemistry; D020930:MAP Kinase Kinase Kinases / Q000037:antagonists & inhibitors*; D051379:Mice; D008810:Mice, Inbred C57BL; D011725:Pyridines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D060888:Real-Time Polymerase Chain Reaction; D059467:Transcriptome
chemical_list: D001549:Benzamides; C473711:Chir 99021; D011725:Pyridines; D011743:Pyrimidines; C506614:mirdametinib; D004159:Diphenylamine; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; D020930:MAP Kinase Kinase Kinases; D038362:Glycogen Synthase Kinase 3
keywords: GSK3β; MEK; embryonic stem cells; genetic backgrounds