Analysis of in vivo mutation in the hprt and tk genes of mouse lymphocytes.
Determining mutant frequencies in endogenous reporter genes is a tool for identifying potentially genotoxic environmental agents and discovering phenotypes prone to genomic instability and diseases, such as cancer. Here we describe a high-throughput method for identifying mouse spleen lymphocytes having mutations in the endogenous X-linked hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene and the endogenous autosomal thymidine kinase (Tk) gene. The selective expansion of mutant lymphocytes is based on the phenotypic properties of Hprt- and Tk-deficient cells. The same procedure can be utilized for quantitating Hprt mutations in most strains of mice (and, with minor changes, in other mammalian species), whereas mutations in the Tk gene can be determined only in transgenic mice that are heterozygous for inactivation of this gene. Expanded mutants can be further used to classify the types of mutations in the Tk gene (small intragenic mutations vs large chromosomal mutations) and to determine the nature of intragenic mutation in both the Hprt and Tk genes.
Dobrovolsky
Vasily N
VN
Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, US Food and Drug Administration, Jefferson, AR, USA.
Shaddock
Joseph G
JG
Heflich
Robert H
RH
D000818:Animals; D004247:DNA / Q000032:analysis; D004252:DNA Mutational Analysis / Q000379:methods*; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D008214:Lymphocytes / Q000737:chemistry / Q000648:ultrastructure*; D051379:Mice; D008822:Mice, Transgenic; D009154:Mutation; D013937:Thymidine Kinase / Q000235:genetics*
D004247:DNA; D007041:Hypoxanthine Phosphoribosyltransferase; D013937:Thymidine Kinase