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Analysis and Selection for gene-amplified cell based on FISH

Research Project

Project/Area Number 11650817
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field 生物・生体工学
Research InstitutionOsaka University

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)

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)
Project Period (FY) 1999 – 2001
Project Status Completed (Fiscal Year 2001)
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)
Keywordsgene-amplification / FISH / F-MTX / flow cytometer / FISH (Fluore scence in situ hybridization) / dhtr / FISH(Fluorescene in situ hybridization) / FISH
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

Report

(4 results)
  • 2001 Annual Research Report   Final Research Report Summary
  • 2000 Annual Research Report
  • 1999 Annual Research Report
  • Research Products

    (11 results)

All Other

All Publications (11 results)

  • [Publications] T.Yoshikawa, F.Nakanishi, Y.Ogura, D.Oi, T.Omasa, Y.Katakura, M, Kisimoto, K.Suga: "Amplified gene location in chromosomal DNA affected recombinant protein production and stability of amplified genes"Biotechnology Progress. 16. 710-715 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] T.Yoshikawa, F.Nakanishi, S.Itami, D.Kameoka, T.Omasa, Y.Katakura, M.Kishimoto, K.Suga: "Evaluation of stable and highly productive gene amplified CHO cell line based on thelocation of amplified gene"Cytotechnology. 33. 37-46 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] T.Yoshikawa, E.Nakanishi, Y.Ogura, D.Oi, T.Omasa, Y.Katakura, M.Kishimoto, K.Suga: ""Flow cytometry : An improved method for the selection of highly productive gene-amplified CHO cells using flow cytometry""Biotechnol. Bioeng.. 74. 435-442 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] F.Nakanishi, T.Yoshikawa, S.Itami, T.Omasa, Y.Katakura, M.Kishimoto, K.Suga: ""Evaluation of stability in the dhfr gene amplification systemsising fluorescencein situ hybridization""Animal Cell Teclinology : Basic & Applied Aspect. 10. 259-263 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] T. Yoshikawa, F. Nakanishi, S. Itami, D. Kameoka, T. Omasa, Y. Katakura, M. Kishimoto and K. Suga: "Evaluation of stable and highly productive gene amplified CHO cell line based on the location of amplified gene"Cytotechnology. 33. 37-46 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] T. Yoshikawa, F. Nakanishi, Y. Ogura, D. Oi, T. Omasa, Y. Katakura, M. Kishimoto and K. Suga: "Amplified gene location in chromosomal DNA affected recombinant protein production and stability of amplified genes"Biotechnology Progress. 16. 710-715 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] T. Yoshikawa, F. Nakanishi, Y. Ogura, D. Oi, T. Omasa, Y. Katakura, M. Kishimoto, and K. Suga: "Flow cytometry: An improved method for the selection of highly productive gene-amplified CHO cells using flow cytometry"Biotechnol. Bioeng.. 74. 435-442 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] F. Nakanishi, T. Yoshikawa, S. Itami, T. Omasa, Y. Katakura, M. Kishimoto and K. Suga: "Evaluation of stability in the dhfr gene amplification system using fluorescence in situ hybridization"Animal Cell Technology; Basic & Applied Aspect. 10. 259-263 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] T.Yoshikawa, E.Nakanishi, Y.Ogura, D.Oi, T.Omasa, Y.Katakura, M.Kishimoto, K.Suga: "Flow cytometry : An improved method for the selection of highly productive gene-amplified CHO cells using flow cytometry"Biotechnology and Bioengineering. 74. 435-442 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Tomohiro Yoshikawa,Fumi Nakanishi,Seima Itami,Daisuke Kameoka,Takeshi Omasa,Yoshio Katakura,Michumasa Kishimoto and Ken-ichi Suga: "Evaluation of stable and highly productive gene amplified CHO cell line based on the location of amplified gene"Cytotechnology. Vol33 No.S,1-3. 37-46 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Tomohiro Yoshikawa,Fumi Nakanishi,Yuki Ogura,Daisuke Oi,Takeshi Omasa,Yoshio Katakura,Michumasa Kishimoto and Kan-ichi Suga: "Amplified gene location in chromosomal DNA affected recombinant protein production and stability of amplified genes"Biotechnology Progress. Vol.16 No.5. 710-715 (2000)

    • Related Report
      2000 Annual Research Report

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Published: 1999-04-01   Modified: 2016-04-21  

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