Establishment of Hypoxanthine Phosphoribosyl-transferase(HPRT)-locus Mutation Assay System in Mouse ES Cells

Abstract

A hypoxanthine phosphoribosyltransferase (HPRT)-locus mutation assay system in mouse ES cells was established and several typical mutagens/carcinogens were examined. The results were as follows: 1) the frequency of spontaneous mutation was 1 per 10⁶ cells, 2) direct-acting mutagens: N-methyl-N'- nitro-N-nitrosoguanidine (0.5-2µg/ml) and ethyl methane sulfonate (200-600µg/ml) respectively induced 30-150 and 6-70 mutants per 10⁶ cells, 3) mutagens activated by ES cells: 4 nitroquino-line-1-oxide (0.5, 1µM) induced 20-50 mutants per 10⁶ cells; mitomycinC, even at a highly toxic dose (0.25µg/ml), did not induce mutation, 4) ES cells seemed not to have metabolic activation system for polycyclic aromatic hydrocarbons (PAHs) but 7,12-dimethylbenz(a)anthracene(0.5-2µM) induced 20-80 mutants per 10⁶ cells when added to ES cells on a feeder layer. Thus the frequencies of ES cell-mutation (spontaneous and induced) were approximately one tenth those of V79 cells, and 5) the degree of metabolic cooperation of the ES cells was almost the same as that reported for V79 cells. Thus feeder cells (unless 6TG-resistant feeder cells) should not be used and the cell number should be under 5 x 10⁵ cells per 10 cm dish when 6TG selection is started.