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2001 Fiscal Year Final Research Report Summary

Development of new method for construction of adenovirus vector using gene exchange reaction

Research Project

Project/Area Number 11470076
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Virology
Research InstitutionThe University of Tokyo

Principal Investigator

KANEGAE Yumi  Institute of Medical Science, The University of Tokyo, Research Instructor, 医科学研究所, 助手 (80251453)

Co-Investigator(Kenkyū-buntansha) SAITO Izumu  Institute of Medical Science, The University of Tokyo, Professor, 医科学研究所, 教授 (70158913)
Project Period (FY) 1999 – 2000
KeywordsAdenovirus vector / Cre / loxP / Gene exchange
Research Abstract

To develop a method for constructing adenovirus vector using gene exchange reaction mediated by Cre recombination. We assayed mutant loxPs recognized independently of wild-type loxP, authentic target sequences of Cre, and identified loxP V as a mutant loxP with high sensitivity and specificity. We examined efficiency of gene exchange reaction using both wild-type loxP and mutant loxP V and showed that the efficiency was no less than 10 %.
The recipient virus for constructing recombinant adenovirus of El -deleted type was designed so that the viral packaging signal (Ψ) was able to be excised by Cre-mediated recombination between a pair of loxP sites located at the both sides of Ψ sequences. We introduced to Cre-expressing 293 cells both a recipient virus and a donor plasmid containing Ψ sequences and a purpose gene flanked with loxP and V. After five cycles of infection of Cre-293 cells with crude viral stock obtained, an adenovirus vector expressing the purpose gene was generated through gene exchange reaction. Pure purpose virus vector was able to be easily isolated after limited dilution using 293 cells.
To construct "gutted vector" whose all viral genes were replaced by a purpose DNA, we prepared a new recipient virus that serves as a helper virus. We also constructed four different donor cosmid cassettes containing cleavage sites of 8-base recognition enzyme as a cloning site in order to insert a purpose gene and a stuffer DNA of large size.

  • Research Products

    (8 results)

All Other

All Publications (8 results)

  • [Publications] Miyazaki, T. et al.: "Reciprocal role of ERK and NF-kappaB pathways in survival and activation of osteoclasts"J. Cell Biol.. 148. 333-342 (2000)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Wakita, T. et al.: "Possible role of cytotoxic T cells in acute liver injury in hepatitis C virus cDNA transgenic mice mediated by Cre/loxP system"J. Med Virol.. 62. 308-317 (2000)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Sakai, Y. et al.: "Gene therapy for hepatocellular carcinoma using two recombinant adenovirus vectors with alpha-fetoprotein promoter and Cre/lox P system"J. Virol. Method. 92. 5-17 (2001)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Nakano, M. et al.: "Efficient gene activation in cultured mammalian cells mediated by FLP recombinase-expressing recombinant adenovirus"Nucl. Acids Res.. 29. e40 (2001)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Miyazaki, T. et al: "Reciprocal role of ERK and NF-kappaB pathways in survival and activation of osteoclasts"J Cell Biol. 148. 333-342 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Wakita, T. et al: "Possible role of cytotoxic T cells in acute liver injury in hepatitis C virus CDNA transgenic mice mediated by Cre/loxP system"J Med Virol. 62. 308-317 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Sakai, Y. et al: "Gene therapy for hepatocellular carcinoma using two recombinant adenovirus vectors with α-fetoprotein promorter and Cre/loxP system"J Virol Methods. 92. 5-17 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Nakano, M. et al: "Efficient gene activation in cultured mammalian cells mediated by FLP recombinase-expressing recombinant adenovirus"Nucleic Acids Res. 29. e40 (2001)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 2003-09-17  

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