Open AccessDevelopment of a universal gap repair vector for yeast-based screening of knockout rodents
Author(s) -
Kai-Shun Chen,
Michael N. Gould
Publication year - 2004
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/04373st02
Subject(s) - gene knockout , mutagenesis , biology , gene knockin , genetics , shuttle vector , computational biology , dna repair , gene , knockout mouse , vector (molecular biology) , mutation , recombinant dna
Recently, we reported the production of the first knockout rats by combining N-ethyl-Nnitrosourea (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 × Apc Min/+ )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.