In vitro induction, expression and selection of thioguanine-resistant mutants with human T-lymphocytes

Conditions have been defined to measure the in vitro induction of mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in human T-lymphocytes by a cell cloning assay. The in vitro growth of mass cultures as well as cell cloning is accomplished by the use of crude T-cell growth factor (TCGF) and irradiated human lymphoblastoid feeder cells. These initial studies employed irradiation of G0 phase peripheral blood mononuclear cells from a single individual. After exposure to gamma-irradiation from a 137Cs source, the cells were stimulated with the mitogen phytohemagglutinin (PHA) and maintained in exponential growth with exogenous TCGF to allow phenotypic expression of the 6-thioguanine-resistant (TGr) mutants. The mutant frequency was determined by measuring cell cloning efficiency in microtiter dishes in the absence and presence of TG, with an optimal selection density of 1 X 10(4) cells/well. The development of this in vitro assay should allow direct study of susceptibility to gamma-irradiation in the human population in terms of both cytotoxicity and mutation induction.