Induction and Selection for Loss of function Mutations

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DESCRIPTION (provided by applicant): The goal of this proposed project is to develop a technology to produce rats with germ line modifications which result in the loss of function of specific selected genes (knockouts). Methods will be developed to efficiently mutagenize male rats with ENU and then breed these rats to non-mutagenized females to produce F1 pups. These pups will then be screened before weaning for loss-of-function mutations in selected genes using efficient yeast-based assays. This will be accomplished through the following aims: protocols for mutagenizing rat strains with ENU will be established. Mutagenesis protocols will be evaluated by quantifying the frequency of pups with visible phenotypic mutations (phenodeviants) and by quantifying specific locus mutations in male germ cells using the rat Big Blue assay. Positive control models for the assays will be established. The initial models will be rat knockout strains (e.g., Brca2 knockout) which have a functional mutation but modest nonsense-mediated RNA decay (NMD). A second rat model will be used to evaluate strategies to minimize NMD. Rats with mutations in the agouti gene, having a high level of NMD of mutant agouti RNA, will be used for this purpose. Strategies to minimize NMD include: yeast assay optimization, pharmacological interventions, and genetic approaches. A series of three yeast-based assays to efficiently and economically identify mutagenized rats carrying functional mutations in selected target genes will be used. The first is referred to as the "functional protein assay." In this assay, the investigators will select for functional mutations by screening for a gene product that has full or partial loss of protein function. The second assay type is referred to as the "cDNA truncation" assay, and the third is the "genomic DNA (gDNA) truncation" assay. These truncation assays screen for mutations that interfere with protein translation. Knockout protocols will continuously be evaluated for their ability to provide knockout rats. Ninety rats per week will be screened for knockouts of approximately ten genes or gene fragments. This assay will be modified at specific intervals to reflect the investigator's improving technologies. Establishing knockout technologies in the rat will provide the means to develop unique models that will extend knowledge of the genetics underlying human diseases and aid in the development of novel drugs to both prevent and treat these diseases.