| Project/Area Number |
11650817
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
生物・生体工学
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| Research Institution | Osaka University |
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Principal Investigator |
OMASA Takeshi Department of Biotechnology, Grad. Sch. of Eng. Osaka Univ. Assistant Prof., 大学院・工学研究科, 助手 (00252586)
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| Co-Investigator(Kenkyū-buntansha) |
KATAKURA Yoshio Dept. of Biotech., Grad. Sch. of Eng., Osaka Univ. Assistant Prof., 大学院・工学研究科, 助手 (50263207)
KISHIMOTO Michimasa Dept. of Biotech., Grad. Sch. of Eng., Osaka Univ. Associate Prof., 大学院・工学研究科, 助教授 (00144436)
菅 健一 大阪大学, 大学院・工学研究科, 教授 (20029250)
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| Project Period (FY) |
1999 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2001: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2000: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1999: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Keywords | gene-amplification / FISH / F-MTX / flow cytometer / FISH (Fluore scence in situ hybridization) / dhtr / FISH(Fluorescene in situ hybridization) / FISH |
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| Research Abstract |
In order to establish an easy and quick construction method for obtaining a stable and highly productive gene-amplified recombinant Chinese Hamster Ovary (CHO) cell line, various kinds of stepwise methotrexate (MTX) selection were carried out. The specific growth and production rates of the cell were compared with each other, and the distribution of the amplified gene location was determined using fluorescence in situ hybridization (FISH). The specific growth and production rates of the cell pool reached the highest levels under the selection condition in which the stepwise increase in the MTX concentration was most gradual; about 82 % of amplified genes were observed near the telomeric region. To clarify the behavior of gene-amplified cell pools during a stepwise increase of MTX concentration, we investigated isolated gene-amplified clones derived from gene amplified cell pools. As a result, telomere-type clones, in which the amplified gene was located near the telomeric region, were
… More
found to be more stable and productive the other types of clones. Telomere-type clones had over 100 copies of amplified genes in the chromosomal DNA In contrast a large number of other types of clones had less than 10 copies of amplified genes. During long-term cultivation in the absence of MTX, in other types of clones, amplified genes rapidly decreased in the chromosomal DNA.
Using a fluorescein isothiocyanatelabeled methotrexate (F-MTX) reagent with flow cytomery, we were easily able to distinguish between highly productive cells and the other types of cells. The level of fluorescence differed according to the difference in resistance to MTX. Based on this new finding, highly productive gene-amplified cells could be isolated from heterogeneous gene amplified cell pools more easily than by the method of limiting dilution assay. The limiting-dilution method requires several months to obtain highly productive gene amplified cells, while our flowcytometry-based method of selection requires only a few weeks. Less
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