Development of a universal gap repair vector for yeast-based screening of knockout rodents.
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.
University of Wisconsin Medical School, Madison, WI, USA.
Kai-Shun
KS
Chen
Michael N
MN
Gould
D025601:Adenomatous Polyposis Coli Protein / Q000172:deficiency* / Q000235:genetics; D000818:Animals; D030801:Animals, Genetically Modified; D019313:BRCA1 Protein / Q000172:deficiency* / Q000235:genetics; D024682:BRCA2 Protein / Q000172:deficiency* / Q000235:genetics; D002478:Cells, Cultured; D004252:DNA Mutational Analysis; D018076:DNA, Complementary; D018390:Gene Targeting / Q000379:methods*; D005820:Genetic Testing; D005822:Genetic Vectors; D051379:Mice; D018345:Mice, Knockout; D016133:Polymerase Chain Reaction; D051381:Rats; D017207:Rats, Sprague-Dawley / Q000235:genetics*; D015003:Yeasts / Q000235:genetics*
D025601:Adenomatous Polyposis Coli Protein; D019313:BRCA1 Protein; D024682:BRCA2 Protein; D018076:DNA, Complementary