Production of mouse ES cells homozygous for Cdk5-phosphorylated site mutation in c-Src alleles

J Biochem. 2003 May;133(5):563-9. doi: 10.1093/jb/mvg072.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • Amino Acid Substitution
  • Animals
  • Cyclin-Dependent Kinase 5
  • Cyclin-Dependent Kinases / genetics*
  • Embryo, Mammalian / cytology*
  • Gene Targeting / methods
  • Genes, src / genetics*
  • Genetic Vectors
  • Homozygote
  • Immunoblotting
  • Mice
  • Peptide Mapping / methods
  • Phosphopeptides / analysis
  • Phosphorylation
  • Point Mutation
  • Recombination, Genetic / genetics
  • Stem Cells / enzymology
  • Stem Cells / physiology*
  • Transfection


  • Phosphopeptides
  • Cyclin-Dependent Kinase 5
  • Cdk5 protein, mouse
  • Cyclin-Dependent Kinases