Introduction of a point mutation into the mouse genome by homologous recombination in embryonic stem cells using a replacement type vector with a selectable marker

Nucleic Acids Res. 1993 Jun 11;21(11):2613-7. doi: 10.1093/nar/21.11.2613.


The introduction of small mutations instead of null alleles into the mouse genome has broad applications to the study of protein structure-function relationships and the creation of animal models of human genetic diseases. To test a simple mutational strategy we designed a targeting vector for the mouse proopiomelanocortin (POMC) gene containing a single nucleotide insertion that converts the initial tyrosine codon of beta-endorphin 1-31 to a premature translational termination codon and introduces a unique Hpal endonuclease restriction site. The targeting vector also contains a neo cassette immediately 3' to the last POMC exon and a herpes simplex virus thymidine kinase cassette to allow positive and negative selection. Homologous recombination occurred at a frequency of 1/30 clones of electroporated embryonic stem cells selected in G418 and gancyclovir. 10/11 clones identified initially by a polymerase chain reaction (PCR) strategy had the predicted structure without evidence of concatemer formation by Southern blot analysis. We used a combination of Hpa I digestion of PCR amplified fragments and direct nucleotide sequencing to further confirm that the point mutation was retained in 9/10 clones. The POMC gene was transcriptionally silent in embryonic stem cells and the targeted allele was not activated by the downstream phosphoglycerate kinase-1 promoter that transcribed the neo gene. Under the electroporation conditions used, we have demonstrated that a point mutation can be introduced with high efficiency and precision into the POMC gene using a replacement type vector containing a retained selectable marker without affecting expression of the allele in the embryonic stem cells. A similar strategy may be useful for a wide range of genes.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alleles
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Blotting, Southern
  • DNA / isolation & purification
  • Embryo, Mammalian
  • Exons
  • Ganciclovir / pharmacology
  • Genetic Markers
  • Genetic Vectors
  • Genome
  • Gentamicins / pharmacology
  • Mice
  • Molecular Sequence Data
  • Mutagenesis, Insertional
  • Oligodeoxyribonucleotides
  • Point Mutation*
  • Polymerase Chain Reaction
  • Pro-Opiomelanocortin / genetics*
  • Protein Biosynthesis
  • Recombination, Genetic*
  • Simplexvirus / enzymology
  • Simplexvirus / genetics
  • Stem Cells / drug effects
  • Stem Cells / metabolism*
  • Thymidine Kinase / genetics
  • Thymidine Kinase / metabolism
  • Tyrosine
  • beta-Endorphin / genetics*


  • Genetic Markers
  • Gentamicins
  • Oligodeoxyribonucleotides
  • Tyrosine
  • beta-Endorphin
  • Pro-Opiomelanocortin
  • DNA
  • antibiotic G 418
  • Thymidine Kinase
  • Ganciclovir