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 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 x 10(5) CFU/microgram of genomic DNA) of the shuttle vector and spontaneous mutation frequency (3.9 x 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 transversions 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.
Yamada
T
T
Environmental and Health Science Laboratory, Sumitomo Chemical Co., Ltd., Osaka, Japan. yamadat9@sc.sumitomo-chem.co.jp
Odawara
K
K
Kishida
F
F
Nakatsuka
I
I
Yoshitake
A
A
D000410:Alanine Transaminase / Q000235:genetics; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D002460:Cell Line; D002869:Chromosome Aberrations; D006224:Cricetinae; D003412:Cricetulus / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005822:Genetic Vectors; D007621:Karyotyping; D008168:Lung / Q000166:cytology; D008769:Methylnitronitrosoguanidine / Q000633:toxicity; D016296:Mutagenesis / Q000187:drug effects*; D009152:Mutagenicity Tests / Q000379:methods; D010430:Pentosyltransferases; D017354:Point Mutation / Q000187:drug effects*; D011506:Proteins; D014162:Transfection
D001426:Bacterial Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D011506:Proteins; D008769:Methylnitronitrosoguanidine; D010430:Pentosyltransferases; C071962:Gpt protein, E coli; D000410:Alanine Transaminase