| Project/Area Number |
07557029
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 試験 |
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| Research Field |
Virology
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| Research Institution | Hokkaido University |
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Principal Investigator |
TAKADA Kenzo Hokkaido University School of Medicine, Cancer Institute, Department of Virology, Professor, 医学部, 教授 (30133721)
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| Co-Investigator(Kenkyū-buntansha) |
AKITAYA Tatsuo Pharmaceutical Research Laboratory Hitach Chemical Co., Ltd., Research Associate, 医薬品研究所, 研究員
SUGIURA Makoto Hokkaido University School of Medicine, Cancer Institute, Department of Virology, 医学部, 助手 (20241317)
秋田谷 龍雄 日立化成工業株式会社, 医薬品研究所, 研究員
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| Project Period (FY) |
1995 – 1996
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| Project Status |
Completed (Fiscal Year 1996)
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| Budget Amount *help |
¥9,000,000 (Direct Cost: ¥9,000,000)
Fiscal Year 1996: ¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 1995: ¥6,000,000 (Direct Cost: ¥6,000,000)
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| Keywords | EB virus / Viral vector / Gene therapy / ウイルススペクター |
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| Research Abstract |
We lack a host cell supporting an efficient replication of Epstein-Barr virus (EBV). Recently, we isolated EBV-negative cell clones from the Akata cell line (referred as Akata-) , and found that Akata-cells are good hosts for EBV propagation and are suitable for propagation of recombinant EBV clonally, which becomes a powerful tool for determining EBV genetics and which makes it possible to use EBV as a vector for gene therapy. The procedure for producing recombinant EBV is as follows. EBV-positive Akata cells have about 20 copies of EBV plasmid per cell. After insertion of the drug-resistant gene into an EBV plasmid of EBV-positive Akita cells by homologous recombination, a virus preparation produced, a mixture of wild-type EBV and recombinant EBV is used to infect EBV-negative Akata cells. After 3 weeks of incubation in the selective media, many drug-resistant clones are isolated very easily, and most of them are infected with recombinant EBV only. By treatment of cells with anti-Ig antibodies a large amount of recombinant EBV is produced.
For use as a vector for human gene therapy, we need to develop an EBV vector deleted of EBV genes that have potentially oncogenic activities. For that purpose, in the present study, we aimed to generate a packaging cell for EBV propagation. It is known that there is a size limitation for the EBV genome being packaged into virus particle. The genome size of EBV is about 170 kbp, and the genome over 200 kbp is not packaged into virus particles. Therefore, we intended and succeeded to insert a 30 kbp of foreign DNA into one of 20 EBV plasmid in Akata cells. Now we are trying to isolate cell clones that contained a recombinant plasmid only, which should become an ideal host for propagating EBV recombinants deleted of all the transforming genes.
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