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

Hirohisa Tsuda*, Kiyoshi Sasaki and Noriho Tanaka

Hatano Research Institute, Food and Drug Safety Center, 729-5, Ochiai, Hadano, Kanagawa 257-8523, Japan

AATEX 11(2):118-128, 2005

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.

Key words:HPRT Locus mutation, ES cell, metabolic cooporation

* Present Address: Intellectual Property Office (Honjo Liaison Office), Kumamoto University, 1-1-1, Honjo, Kumamoto City, Kumamoto 862-8556, Japan