Methods for detecting somatic mutations in vitro: the human T-cell cloning assay selecting for HPRT mutants.

Abstract

The T-cell cloning assay, which detects mutations in the gene for hypoxanthine-guanine phosphoribosyltransferase (HPRT), is the most well-developed reporter system for studying specific locus mutation in human somatic cells. The assay is based on a mitogen- and growth factor-dependent clonal expansion of peripheral T lymphocytes in which the 6-thioguanine-resistant HPRT mutants can be selected, enumerated, and collected for molecular analysis of their mutational nature. The assay provides a unique tool for studying in vivo and in vitro mutagenesis and for investigating the functional impact of common polymorphisms in metabolism and repair genes. The present chapter presents a simple and reliable method for the enumeration of HPRT mutant frequency induced in vitro without using any source of recombinant interleukin-2. The other main feature is that only truly induced and unique mutants are collected for further analysis.