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Gene transfer methods using mammalian artificial chromosomes

Research Project

Project/Area Number 10044326
Research Category

Grant-in-Aid for Scientific Research (B).

Allocation TypeSingle-year Grants
Section一般
Research Field Molecular biology
Research InstitutionFujita Health University

Principal Investigator

OKAZAKI Tuneko  (Institute for comprehensive medical science, Fujita Health University Professor), 総合医科学研究所, 教授 (10022584)

Co-Investigator(Kenkyū-buntansha) MASASHI Ikeno  Fujita health university, Research Associate, 総合医科学研究所, 助手 (80298546)
MITANI Kohno  カリフォルニア大学ロサンゼルス校, 医学部, 助教授
HIETER Phill  プリティッシュコロンビア大学, 医学部CMMT研究所, 教授
Project Period (FY) 1998 – 2000
Project Status Completed (Fiscal Year 2000)
Budget Amount *help
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2000: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1999: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1998: ¥1,500,000 (Direct Cost: ¥1,500,000)
KeywordsHAC / Cre / loxP / Alphoid sequence / Microcell-mediated chromosome transfer / Ad / HAC vector / lox / 微小核融合法 / テロメア配列 / MAC / HACペクター
Research Abstract

Our research objective is the development of basic technologies to utilize mammalian artificial chromosomes for gene delivery vectors and we collaborated with Prof. P.Hieter at Columbia University and Prof. K.Mitani at California University to proceed the research. Okazaki's group challenged the construction of HACs in which a YAC clone containing a large human genomic area was used as a starting material. The arms of the YAC clone were modified with human telomere sequences, loxP sequence and mammalian selection marker. To insert alphoid sequence into the modified YAC by Cre/loxP recombination system, a BAC carrying 100kb alphoid sequence and loxP sequence was constructed. In order to generate HACs, the method of the YAC purification free from yeast genomic DNA was crucial and we obtained useful information through discussion with Prof. Hieter. In order to use this HACs as gene delivery vectors in the other cell lines, we transferred HACs, which had been formed in the HT1080 cell line, into mouse A9 cells by microcell-mediated chromosome transfer (MMCT) method. We confirmed that HACs transferred into the A9 cells were stably maintained. Mitani's group in collaboration with our group succeeded in the construction of Ad/HAC vector containing human telomere sequences and alphoid sequence in a helper-dependent adenovirus vector. When this DNA was introduced into HT1080 cells by lipofection, transformants were obtained. We are currently analyzing the HAC formation in the transformants. It is important future subject whether HAC is formed efficiently from Ad/HAC vectors when infected to cells as virus particles.

Report

(4 results)
  • 2000 Annual Research Report   Final Research Report Summary
  • 1999 Annual Research Report
  • 1998 Annual Research Report
  • Research Products

    (15 results)

All Other

All Publications (15 results)

  • [Publications] K.Yoda,T.Okazaki, et.al: "In vitro assembly of the CENP-B/alpha-satellite DNA/core histone complex : CENP-B causes nucleosome positioning."Genes to cells. 3. 533-548 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] H.Masumoto,M.Ikeno,T.Okazaki, et.al: "Assay of centromere function using a human artificial chromosome."Chromosoma. 107. 406-416 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] K.Yoda,T.Okazaki, et.al: "Human centromere protein A (CENP-A) can replace histone H3 in nucleosome reconstitution in vitro."Proc.Nath.Acod.Sci.USA. 97. 7266-7271 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] K.Yoda, T.Okazaki, et.al: "In vitro assembly of the CENP-B/alpha-satellite DNA/core histone complex : CENP-B causes nucleosome positioning."Genes to cells. 3. 533-548 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] H.Masumoto, M.Ikeno, T.Okazaki, et.al: "Assay of centoromere function using a human artificial chromosome."Chromosoma. 107. 406-416 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] K.Yoda, T.Okazaki, et.al: "Human centoromere protein A (CENP-A) can replace histone H3 in nucleosome reconstitution in vitro."Proc.Nath.Acod.Sci.USA. 97. 7266-7271 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] K.Yoda,T.Okazaki: et.al: "Human centromere protein A (CENP-A) can replace histone H3 in nucleosome reconstitution in vitro."Proc.Nath.Acod.Sci.USA. 97. 7266-7271 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] K.Yamada,M.Ikeno,T.Okazaki: et.al: "Genomic Organization, chromosomal localization, and the complete 22kb DNA sequence of the human GCMa/GCM1, a placenta-specific transcription factor gene."Biochem.Biophys.Res.Commun.. 278. 134-139 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] K. Yamada, T. Okazaki et al: "A GCM motif protein is involved in placenta-specific expression of human aromatase Gene"J. Biol. Chem. 274(45). 32279-32286 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] 池野正史、岡崎恒子: "哺乳動物の人工染色体"生体の化学. 50(6). 602-611 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] 岡崎恒子: "DNA複製機構の解明 A.コーンバーグ"Kirin Information on Drugs and Science. 9(4). 12-15 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] J.Iwahara: "A helix-turn-helix structure unit in thehuman centromere protein B(CENP-B)" EMBO Journal. 17.3. 827-837 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] M.Ikeno: "Construction of YAC based mammalian artificial chromosomes." Nature Biotech. 16. 431-439 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] K.Yoda: "In vitro assembly of the CENP-B/a-satallite DNA/core histone complex:CENP-B couses uncleosome positioning." Genes to Cells. 3. 533-548 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] H.Masumoto: "Assay of centromere function using a human artificial chromosome." Chromosome. 107. 406-416 (1998)

    • Related Report
      1998 Annual Research Report

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Published: 1998-04-01   Modified: 2016-04-21  

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