| Project/Area Number |
60580167
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
放射線5生物学
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| Research Institution | Osaka University |
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Principal Investigator |
ISHII Yutaka Faculty of Medicine, Osaka University, Assoc. Prof., 医学部, 助手 (20028509)
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| Co-Investigator(Kenkyū-buntansha) |
SHINAGAWA Hideo Research Institute for Microbial Diseases, Osaka University, Assoc. Prof., 微生物病研究所, 助教授 (40029799)
KATO Takesi Faculty of Medicine, Osaka University, Assist. Prof., 医学部, 講師 (90028382)
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| Project Period (FY) |
1985 – 1986
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| Project Status |
Completed (Fiscal Year 1986)
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| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1986: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1985: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | Mutagen Specificity / Shuttle Vector / HPRT / マウス培養細胞 |
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| Research Abstract |
Complementary DNA of human HPRT (hypoxanthine phosphoribosyl transferase) was used as the target DNA for the detection of mutation. This cDNA was inserted into the shuttle vector, pZIP-NeoSV(X)1, which was derived from retrovirus. The vector constructed, pZIP(X)HPRT, which can express HPRT and NEO (neomycin-resistance) genes, was transfected into 2TGOR cells derived from mouse Balb/c 3T3 cell by the following three methods; (a) Ca-phosphate transfection, (b) co-transfection with helper virus DNA (pMOV <psi^-> which is deficient in packaging signal of viral RNA) and (c) infection of defective viruses obtained from the cells constructed by method (b). Transformed cell lines were selected by HAT medium with G418 (derivative of neomycin). Among those, 6 cell lines showed the low frequency of spontaneous mutation to <6TG^R> . Three out of the 6 cell lines showed the mutability induced by ethyl methanesulfonate treatment. Vector DNAs integrated into host cell chromosomes were recovered from transformed cells by the cell fusion with COS cells. Many of them showed deletions and rearrangements, and few were recovered as intact vectors. However, efficient recovery of the intact vector DNAs were shown by the cell line vHPRT-12 which was obtained by the method (c). These intact vector DNAs were used for the analysis of base sequence in HPRTcDNA by synthesized primers and dideoxy method. Six hundred and fifty four base pairs of coding region of HPRTcDNA were coincident with the base sequence of HPRTcDNA which was already reported in papers. Thus the cell line, vHPRT-12 is appeared to be siutable for this project. The spontaneous and X-ray induced <6TG^R> mutants were obtained and intact vector DNAs were recovered from these mutants for the analysis of base sequence.
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