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J. Biochem, 2003, Vol. 133, No. 5 563-569
© 2003 Japanese Biochemical Society

BIOTECHNOLOGY

Production of Mouse ES Cells Homozygous for Cdk5-Phosphorylated Site Mutation in c-src Alleles

Goro Kato+ and Shuichiro Maeda

Department of Biochemistry, Yamanashi Medical University, Nakakoma, Yamanashi 409-3898

c-Src-null mutants have not provided a full understanding of the cellular functions of c-Src, reflecting the functional redundancy among Src family members. c-Src is phosphorylated by cyclin-dependent kinase 1 (Cdk1) and Cdk5 at Ser75 in the unique amino terminal c-Src–specific domain. The specific roles of c-Src may be assessed by establishing mouse embryonic stem (ES) cells homozygous for a point mutation at Ser75. Mammalian homozygous cultured cells with a point mutation, however, have not yet been produced by gene targeting. Here we show an efficient procedure for producing ES cell clones bearing a homozygous Ser75 to Asp mutation in the c-src gene. This procedure was developed by combining two previously reported strategies: our procedure for introducing a point mutation into one allele with no exogenous sequence, and the high-geneticin (G418) selection procedure for introducing a mutation into both alleles. The mutant clones expressed the same levels of c-Src protein and autophosphorylation activity as wild-type cells, but the mutant c-Src was not phosphorylated on Ser75 during mitosis. This procedure is feasible for generating cells homozygous for a subtle mutation in most genes, and is expected to be applicable to other somatic cell lines.

+ To whom correspondence should be addressed. E-mail: gkato{at}res.yamanashi-med.ac.jp


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