Production of mouse ES cells homozygous for Cdk5-phosphorylated site mutation in c-Src alleles.
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.
Kato
Goro
G
Department of Biochemistry, Yamanashi Medical University, Nakakoma, Yamanashi 409-3898, Japan. gkato@res.yamanashi-med.ac.jp
Maeda
Shuichiro
S
D000483:Alleles; D019943:Amino Acid Substitution; D000818:Animals; D051360:Cyclin-Dependent Kinase 5; D018844:Cyclin-Dependent Kinases / Q000235:genetics*; D004622:Embryo, Mammalian / Q000166:cytology*; D018390:Gene Targeting / Q000379:methods; D016391:Genes, src / Q000235:genetics*; D005822:Genetic Vectors; D006720:Homozygote; D015151:Immunoblotting; D051379:Mice; D010449:Peptide Mapping / Q000379:methods; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D017354:Point Mutation; D011995:Recombination, Genetic / Q000235:genetics; D013234:Stem Cells / Q000201:enzymology / Q000502:physiology*; D014162:Transfection
D010748:Phosphopeptides; D051360:Cyclin-Dependent Kinase 5; C495906:Cdk5 protein, mouse; D018844:Cyclin-Dependent Kinases