Genotype-based screen for ENU-induced mutations in mouse embryonic stem cells

Nat Genet. 2000 Mar;24(3):314-7. doi: 10.1038/73557.

Abstract

The ability to generate mutations is a prerequisite to functional genetic analysis. Despite a long history of using mice as a model system for genetic analysis, the scientific community has not generated a comprehensive collection of multiple alleles for most mouse genes. The chemical mutagen of choice for mouse has been N-ethyl-N-nitrosourea (ENU), an alkylating agent that mainly causes base substitutions in DNA, and therefore allows for recovery of complete and partial loss-, as well as gain-, of-function alleles . Specific locus tests designed to detect recessive mutations showed that ENU is the most efficient mutagen in mouse with an approximate mutation rate of 1 in 1,000 gametes. In fact, several genome-wide and region-specific screens based on phenotypes have been carried out. The anticipation of the completion of the human and mouse genome projects, however, now emphasizes genotype-driven genetics--from sequence to mutants. To take advantage of the mutagenicity of ENU and its ability to create allelic series of mutations, we have developed a complementary approach to generating mutations using mouse embryonic stem (ES) cells. We show that a high mutation frequency can be achieved and that modulating DNA-repair activities can enhance this frequency. The treated cells retain germline competency, thereby rendering this approach applicable for efficient generation of an allelic series of mutations pivotal to a fine-tuned dissection of biological pathways.

Publication types

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

MeSH terms

  • Amino Acid Substitution
  • Animals
  • Chimera / genetics
  • Codon / genetics
  • DNA Damage
  • DNA Repair
  • Ethylnitrosourea / toxicity*
  • Genotype*
  • Hypoxanthine Phosphoribosyltransferase / genetics
  • Mice
  • Mice, Inbred C57BL
  • Mutagenesis*
  • Mutagens / toxicity*
  • Mutation*
  • O(6)-Methylguanine-DNA Methyltransferase / antagonists & inhibitors
  • O(6)-Methylguanine-DNA Methyltransferase / physiology
  • Point Mutation
  • RNA Splicing / genetics
  • Stem Cells / chemistry
  • Stem Cells / drug effects*

Substances

  • Codon
  • Mutagens
  • O(6)-Methylguanine-DNA Methyltransferase
  • Hypoxanthine Phosphoribosyltransferase
  • Ethylnitrosourea