A short-term whole animal toxicity assay will be a more accurate predictor of compound mutagenicity. Current bacterial mutagenic assays are quick, but lack metabolic intermediates required for testing pro- mutagen activation. Tissue culture assays, while convenient and contain additional metabolites and eukaryotic repair functions, lack good correlation with long-term toxicity testing in whole animals. Long-term whole animal toxicity testing is currently the best measure of carcinogenicity of a compound, but is too slow for the initial broad screening of compounds. The increased concerns of human exposure to mutagenic compounds from such common sources as drinking water, food additives and industrial materials outlines the importance of proper mutagen assessment. Our short-term whole animal toxicity assay is designed to increase the speed and quality of these assessments, filling important gaps left by current toxicity assays. We will generate transgenic mice, containing a lacl mutagenic target gene which can be rapidly and efficiently recovered from mouse cells. The lacl target gene cloned into a lambda phage genome will be integrated into a mouse chromosome by transgenic methods. Following extraction of cellular DNA from tissues, the target gene will be recovered by packaging the lambda phage genome with in vitro packaging extracts. The excised phage will be recovered and the lacl gene assayed for mutations by plating on E. coli indicator strains. Base mutations within the target gene will be identified by rapid DNA sequencing utilizing our recently developed lambda ZAP subcloning system. This assay will permit rapid base specific mutation analysis of DNA targets derived from different tissues, different chromosomal integration sites and differential genetic background following exposure of the animal to suspected mutagens.
