Quantification of thioguanine-resistant lymphocytes from mice irradiated in vivo

Abstract

Adult mice were Co-60 gamma irradiated, and 7 months later splenocytes were isolated, cultured in microwells, and the frequency of hprt-deficient mutants was determined by measuring the cloning efficiency in media with 6-thioguanine. The mutant frequency at 2, 4, and 6 Gy was 1.6 x 10(-5), 4.4 x 10(-5), and 12.7 x 10(-5), respectively. The frequency of spontaneous mutants was 2.5 x 10(-6). The effect of metabolic cooperation on the cloning efficiency of thioguanine-resistant T-cells in selective medium was evaluated in co-cultures with wild-type T-cells. We found that the growth of hprt-deficient T-cells is supported in the presence of thioguanine-inactivated wild-type splenocytes up to a cell density of 5 x 10(5) cells per well. When cell density was higher, cell growth was inhibited. Possibilities and limitations of cloned lymphocytes for the analysis of somatic mutations that occur in vivo are discussed.