Use of a T-lymphocyte clonal assay for determining HPRT mutant frequencies in individual rats

Conditions for detection and isolation of HPRT- mutants in cloned rat T-lymphocytes from individual adult Lewis rats were determined. Similar to cloning of human T-cells, best results were obtained with lectin (PHA)-primed T-lymphocytes of rats. High cloning efficiencies, occasionally exceeding 50%, could be obtained when the target cells employed were isolated from cervical lymph nodes. Feeder cells used were splenocytes, irradiated with 40 Gy of X-rays after priming with Con A. Human interleukin-2, present in LAK supernatant, proved to be capable of inducing proliferative activity of rat T-lymphocytes and could replace conditioned medium from primed rat splenocytes. Under the conditions described in this paper, the frequency of mutants in the HPRT gene of T-lymphocytes in Lewis rats was about 80% lower than that found in human T-lymphocytes from adults. The inverse relationship between mutant frequency and cloning efficiency, clearly demonstrated for human data, could not be established for rats. Treatment of rats with N-ethyl-N-nitrosourea, a potent alkylating agent, resulted in a time- and dose-dependent induction of HPRT- mutants, demonstrating the usefulness of this system to study in vivo mutagenesis.