Host: The Japan Radiation Research Society
The rat is an important murine model for radiation research. Through much work the rat genomic toolbox is almost full a key missing tool was the ability to produce knockout rats. In order to approach this problem we developed a unique technology that combined the use of germline mutagenesis with a yeast truncation gap repair assay to select gene-specific knockout rats. We first developed the protocols to efficiently mutagenize the rat genome with ENU. In order to establish knockout rats for specifically chosen genes, we bred ENU mutagenized males to untreated females to produce F1 pups. These pre-weanling pups were screened for functional mutations .This was accomplished using a highly efficient and economical yeast gap-repair truncation assay. PCR product of the targeted gene together with our reporter vector is co-transformed into yeast that do not express ADE2. The yeast through homologous recombination clone the PCR product into the ADE2 reporter plasmid. In scoring the yeast plates a negative has mostly white large colonies and a positive for a knockout allele have half red colonies rat and half white colonies. We have used this technology to knockout two breast cancer suppressor genes, Brca1 and Brca2. Both of these genes are important in the repair of radiation - DNA damage. [J Radiat Res 44:371-372 (2003)]
