Phenotypic expression time of mutagen-induced 6-thioguanine resistance in Chinese hamster ovary cells (CHO/HGPRT system).

Mutation induction at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells (referred to as the CHO/HGPRT system) can be quantitated by selection for the phenotype of resistance to 6-thioguanine (TG) under stringently defined conditions. The phenotypic expression time, that is, the time interval after mutagen treatment which is necessary befor all mutant cells are able to express the TG-resistant phenotype, has been found to be 7–9 days in this CHO/HGPRT system when the cells are subcultured every 48 h. Subculture in medium with or without hypoxanthine (HX) utilizing trypsin, ethylenediaminetetraacetic acid (EDTA), or ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) for cell removal yields identical results. When subculture at intervals greater than 48 h is employed, a slight lengthening of the expression time is observed. An alternative method to regular subculture has also been achieved by maintaining the cells in a viable, non-dividing state in serum-free medium. This procedure yields a similar time course of phenotypic expression and thus shows that continued cell division is not essential to this expression process. In addition, this observation offers methodology which can significantly reduce the investment of time and money for mutation induction determinations in this mammalian cell gene mutation assay.