Development of a universal gap repair vector for yeast-based screening of knockout rodents

Biotechniques. 2004 Sep;37(3):383-8. doi: 10.2144/04373ST02.

Abstract

Recently, we reported the production of the first knockout rats by combining N-ethyl-N-nitrosourea (ENU)-induced mutagenesis with a yeast-based truncation screening method. To make this new knockout technology more applicable for other laboratories and for high-throughput applications, we have developed a universal gap repair vector that is ready for use in screening for gene knockouts without additional engineering. The universal gap repair vector was validated for its application in both cDNA- and genomic DNA-based yeast truncation mutation assays. Breast cancer genes Brca1, Brca2, and Adenomatosis polyposis coli (Apc) genes from N2 rats of Brca1 and Brca2 knockouts and (Atm x ApcMin/+)F1 mice were examined, respectively. The results indicate that the universal gap repair vector we developed, using randomly selected codons as a universal cassette, is equally efficient at identifying truncation mutations as are those gap repair vectors designed specifically for Brca1 and Brca2. The availability of a universal gap repair vector should facilitate the broader screening of knockouts of most genes of many species using the combined approach of ENU-induced mutagenesis and yeast truncation assay.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, P.H.S.
  • Technical Report

MeSH terms

  • Adenomatous Polyposis Coli Protein / deficiency*
  • Adenomatous Polyposis Coli Protein / genetics
  • Animals
  • Animals, Genetically Modified
  • BRCA1 Protein / deficiency*
  • BRCA1 Protein / genetics
  • BRCA2 Protein / deficiency*
  • BRCA2 Protein / genetics
  • Cells, Cultured
  • DNA Mutational Analysis
  • DNA, Complementary
  • Gene Targeting / methods*
  • Genetic Testing
  • Genetic Vectors*
  • Mice
  • Mice, Knockout
  • Polymerase Chain Reaction
  • Rats
  • Rats, Sprague-Dawley / genetics*
  • Yeasts / genetics*

Substances

  • Adenomatous Polyposis Coli Protein
  • BRCA1 Protein
  • BRCA2 Protein
  • DNA, Complementary