| Project/Area Number |
16360410
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biofunction/Bioprocess
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| Research Institution | Nagoya University |
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Principal Investigator |
IIJIMA Shinji Nagoya University, Graduate School of Engineering, Professor, 大学院・工学研究科, 教授 (00168056)
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| Co-Investigator(Kenkyū-buntansha) |
MIYAKE Katsuhide Nagoya University, Ecotopia Science Institute, Associate Professor, エコトピア科学研究所, 助教授 (90252254)
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| Project Period (FY) |
2004 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥15,100,000 (Direct Cost: ¥15,100,000)
Fiscal Year 2005: ¥7,500,000 (Direct Cost: ¥7,500,000)
Fiscal Year 2004: ¥7,600,000 (Direct Cost: ¥7,600,000)
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| Keywords | Integration / Artificial virus / Integrase / YY1 / Q-vector / RNAガンウイルス / 遺伝子治療 / 核移行 |
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| Research Abstract |
In order to improve the integration of artificial vital DNA into host chromosome, we have studied the components of preintegration complex (PIC) of molony murine leukemia virus as a model system. We found that integrase which is one of the main components of the PIC, physically interacted with a transcription factor YY1. We also found that Bmi-1 which is a subunit of polycomb group repressive complex, interacted with INI-1(integrase interacter-1). INI-1 associates with integrase and is also a main component of PIC. To clarify the interaction between integrase and YY-1, first, we analyzed the interaction by GST-pull down assay. We found that the N-terminal region of YY-1 interacted with the integrase. Because N-terminal region of YY-1 is known as an activation domain and the C-terminal region is a domain for DNA interaction, we assumed that binary binding trough integrase and YY-1 to virus cDNA may affect the integration reaction. Integrase from HIV-1 also interacted with YY-1 by the GST-pull down assay.
In order to improve the viral vector production, we applied a transient viral production system, so-calle d Q-vector system, to molony leukemia virus based vector system. So far now, high titer viral vector preparation has been prepared by so-called packaging cell line method. But the establishment of packaging cell line is laborious and it take longer time. On the other hand, virus vector can be produced within 2-3 days by the Q-vector system. We found that the virus titer produced by the Q-vector was largely dependent on vector size but the titer was comparable to the original packaging cell method if the transgene size was around 2 kb. When the transgene has a cytotoxic effect on animal cells, the Q-vector system gave a higher titer comparing to the packaging cell method.
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