Development of a Universal Gap Repair Vector for Yeast-Based Screening of Knockout Rodents

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.

Related Papers

• → Singlet Oxygen Induces Predominantly G to T Transversions on a Single-Stranded Shuttle Vector Replicated in Monkey Cells(1994)20 cited
• → Studies on direct and indirect effects of DNA damage on mutagenesis in monkey cells using an SV40-based shuttle vector(1989)10 cited
• → The development of transient SV40 based shuttle vectors for mutagenesis studies: Problems and solutions(1989)34 cited
• Molecular analysis of mutations induced by chemical carcinogens in mammalian cells. II. The use of recombinant DNA vectors.(1994)
• From Phenotype to Function:Analysis of Phenotype of p38α Knockout Mice(2002)