A short term whole animal toxicity assay will be a more accurate predictor of compound mutagenicity. Current bacterial mutagenic assays are a 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 increases concerns of human composure 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 anality of these assessments, filing important gaps left by current toxicity assays. We will generate transgenic mice, containing a lac I mutagenic target gene which can be rapidly recovered from mouse cells. The lac I 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 lac I gene assayed for mutations by plating on E. coli indicator stains. 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.
| Callahan, J D; Short, J M (1995) Transgenic lambda/lacI mutagenicity assay: statistical determination of sample size. Mutat Res 327:201-8 |
| Provost, G S; Short, J M (1994) Characterization of mutations induced by ethylnitrosourea in seminiferous tubule germ cells of transgenic B6C3F1 mice. Proc Natl Acad Sci U S A 91:6564-8 |
| Kretz, P L; Kohler, S W; Short, J M (1991) Identification and characterization of a gene responsible for inhibiting propagation of methylated DNA sequences in mcrA mcrB1 Escherichia coli strains. J Bacteriol 173:4707-16 |
| Kohler, S W; Provost, G S; Kretz, P L et al. (1990) The use of transgenic mice for short-term, in vivo mutagenicity testing. Genet Anal Tech Appl 7:212-8 |
| Kohler, S W; Provost, G S; Kretz, P L et al. (1990) Development of a short-term, in vivo mutagenesis assay: the effects of methylation on the recovery of a lambda phage shuttle vector from transgenic mice. Nucleic Acids Res 18:3007-13 |
