DEVELOPMENT OF A NOVEL CHL IU CELL-LINE WITH AN INCORPORATED GPT SHUTTLE VECTOR FOR CONCURRENT ANALYSIS OF GENE-MUTATIONS AND CHROMOSOME-ABERRATIONS/

A cosmid shuttle vector containing the target gene of Escherichia coli gpt coding xanthine-guanine phosphoribosyl transferase was constructe d. The shuttle vector was designed to be rescued into the gpt-deficien t Escherichia coli from Chinese hamster CHL/IU cells through an in vit ro packaging method. Mutations occurred at the target gene can be dete cted with a selective agent, 6-thioguanine (6-TG). The shuttle vector was stably transfected into CHL/IU cells to give several cell lines co ntaining copies of the shuttle vector in the chromosomes. Each cell li ne exhibited a characteristic rescue efficiency (0 to 1.9 x 10(5) CFU/ mu g of genomic DNA) of the shuttle vector and spontaneous mutation fr equency (3.9 x 10(-5) to over 10(-2)) at the 6-TG selection. One trans genic cell line (KN63), which showed a higher rescue efficiency and a low spontaneous mutation frequency, was selected and tested for the ab ility to respond to a genotoxic agent, N-methyl-N'-nitro-N-nitrosoguan idine (MNNG). MNNG increased both the mutation frequency at the target gene and the number of the cells with chromosome aberrations. DNA seq uence analysis of 6-TG mutants showed that predominant mutations (10/1 4) were identified as G:C to A:T transitions in MNNG-induced mutants, whereas transversions were predominant (5/9) in spontaneous mutants. T hese results suggest that this transgenic CHL/IU cell line can be a us eful tool for analyzing the relation between gene mutations and chromo some aberrations. (C) 1997 Elsevier Science B.V.