Development of a novel CHL/IU cell line with an incorporated gpt shuttle vector for concurrent analysis of gene mutations and chromosome aberrations

• Published in: Mutation research. Genetic toxicology and environmental mutagenesis 1997-10, Vol.393 (3), p.295-306
• Main Authors: Yamada, Toru, Odawara, Kyoko, Kishida, Fumio, Nakatsuka, Iwao, Akira Yoshitake
• Format: Article
• Language: English
• Subjects: CHL/IU cell line; gpt; Shuttle vector
• ISSN: 1383-5718; 1879-3592
• DOI: 10.1016/S1383-5718(97)00114-9

A cosmid shuttle vector containing the target gene of Escherichia coli gpt coding xanthine-guanine phosphoribosyl transferase was constructed. The shuttle vector was designed to be rescued into the gpt-deficient Escherichia coli from Chinese hamster CHL/IU cells through an in vitro packaging method. Mutations occurred at the target gene can be detected with a selective agent, 6-thioguanine (6-TG). The shuttle vector was stably transfected into CHL/IU cells to give several cell lines containing copies of the shuttle vector in the chromosomes. Each cell line exhibited a characteristic rescue efficiency (0 to 1.9×10 5 CFU/μg of genomic DNA) of the shuttle vector and spontaneous mutation frequency (3.9×10 −5 to over 10 −2) at the 6-TG selection. One transgenic cell line (KN63), which showed a higher rescue efficiency and a low spontaneous mutation frequency, was selected and tested for the ability to respond to a genotoxic agent, N-methyl- N′-nitro- N-nitrosoguanidine (MNNG). MNNG increased both the mutation frequency at the target gene and the number of the cells with chromosome aberrations. DNA sequence analysis of 6-TG mutants showed that predominant mutations (10/14) were identified as G:C to A:T transitions in MNNG-induced mutants, whereas tranversions were predominant (5/9) in spontaneous mutants. These results suggest that this transgenic CHL/IU cell line can be a useful tool for analyzing the relation between gene mutations and chromosome aberrations.