title: Easy detection of GFP-positive mutants following forward mutations at specific gene locus in cultured human cells.
abstract: We have generated a new mutation assay system using HT1080 human fibrosarcoma cells, which consists of a combination of tetracycline-operator dependent GFP gene (TetO-EGFP) and tetracycline repressor (TetR) genes, where the expression of GFP gene is under strict control of TetR protein, and the TetR gene is located within the endogenous HPRT gene. In this system, any inactivating mutation at the TetR gene or large deletions including the gene itself results in high expression of GFP gene (>200-fold increase) in the cells, which can be readily scored not only by a flow cytometer but also under a fluorescent microscope. With this new cell line, we show that the spontaneous mutation rate at the TetR locus was 2.8-3.4×10(-6)/cell division, slightly lower than the rate at the endogenous HPRT gene of HT1080 cells, and has a dose response to X rays as a mutagen. We also isolated variant clones with elevated spontaneous mutation rate (i.e., genetically unstable cells) following X irradiation. Spontaneous GFP-positive mutants were predominantly base-change mutations at the TetR gene while those obtained after X irradiation often contained large deletions which spanned up to 6Mb. The results indicate that the bacterial TetR/TetO regulatory units work extremely well as a mutation detection system in human cells, and any part of the human genome may be tested for mutation sensitivity following targeted insertion of the TetR gene in a stably expressing gene.
authors.affiliation: Department of Genetics, Radiation Effects Research Foundation, 5-2 Hijiyama Park, Minami-Ku, Hiroshima 732-0815, Japan. asnoda@rerf.or.jp
authors.forename: Asao
authors.initials: A
authors.lastname: Noda
authors.forename: Yuko
authors.initials: Y
authors.lastname: Hirai
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Kodama
authors.forename: Warren W
authors.initials: WW
authors.lastname: Kretzschmar
authors.forename: Kanya
authors.initials: K
authors.lastname: Hamasaki
authors.forename: Yoichiro
authors.initials: Y
authors.lastname: Kusunoki
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Mitani
authors.forename: Harry M
authors.initials: HM
authors.lastname: Cullings
authors.forename: Nori
authors.initials: N
authors.lastname: Nakamura
mesh_terms: D045744:Cell Line, Tumor; D002478:Cells, Cultured; D005354:Fibrosarcoma; D017353:Gene Deletion; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006801:Humans; D009152:Mutagenicity Tests / Q000379:methods*; D009154:Mutation / Q000528:radiation effects*; D009687:Nuclear Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D012097:Repressor Proteins / Q000235:genetics; D012680:Sensitivity and Specificity; D013752:Tetracycline / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics*; D014965:X-Rays
chemical_list: D009687:Nuclear Proteins; D012097:Repressor Proteins; C500210:SAP30BP protein, human; D014157:Transcription Factors; C034811:tetracycline resistance-encoding transposon repressor protein; D049452:Green Fluorescent Proteins; D013752:Tetracycline