Measurement of in vivo mutant frequency in lymphocytes in the mouse

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Abstract
A limiting-dilution cloning technique for quantifying in vivo mutations at the hypoxanthine phosphoribosyl transferase locus in mouse splenocytes was developed. Mouse splenocytes were cultured in round-bottom microwells with irradiated feeder cells, concanavalin A, and a source of interleukin 2 at five cells/well in the absence of thioguanine, and at 5 × 104 cells/well in the presence of 2.5 μg/ml thioguanine; mutant frequency was calculated as the ratio of the cloning efficiencies with or without thioguanine. The geometric mean (95% range) for the mutant frequency in 20 mice was 1.54 × 10−6 (4.7 × 10−7-2.6 × 10−6) and whole-body X-irradiation resulted in a dose-related increase in mutant frequency of up to approximately 20 times the baseline level. The in vivo murine mutation assay should be a useful system for genotoxicity testing and may be of particular value in establishing risk estimates for human populations exposed to genotoxins.