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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
Project Status Completed (Fiscal Year 2001)
Budget Amount *help
¥5,900,000 (Direct Cost: ¥5,900,000)
Fiscal Year 2000: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1999: ¥3,800,000 (Direct Cost: ¥3,800,000)
KeywordsAdenovirus vector / Cre / loxP / Gene exchange / Cre1 loxP
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.

Report

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

    (18 results)

All Other

All Publications (18 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
    • Related Report
      2001 Final Research Report Summary
  • [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
    • Related Report
      2001 Final Research Report Summary
  • [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
    • Related Report
      2001 Final Research Report Summary
  • [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
    • Related Report
      2001 Final Research Report Summary
  • [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
    • Related Report
      2001 Final Research Report Summary
  • [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
    • Related Report
      2001 Final Research Report Summary
  • [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
    • Related Report
      2001 Final Research Report Summary
  • [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
    • Related Report
      2001 Final Research Report Summary
  • [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)

    • Related Report
      2000 Annual Research Report
  • [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.Virot.. 62. 308-317 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Sakai,Y. et.al.: "Gene therapy for hepatocellular carcinoma using two recombinant adenovirus vectors with alpha-fetoprotein promoter and Cre/loxP system."J.Virol.Method. (in press). (2001)

    • Related Report
      2000 Annual Research Report
  • [Publications] Nakan,M. et.al.: "Efficient gene activation in cultured mammalian cells mediated by FLP recombinase-expressing recombinant adenovirus."Nucl.Auds.Res.. (in press). (2001)

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

    • Related Report
      1999 Annual Research Report
  • [Publications] Nagasaki,Y.et al.: "Reversal of hypopigmentation in phenylketonuria mice by adenovirus-mediated gene transfar"Pediatr Res.. 45. 465-473 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Ogasawara,Y.et al.: "Highly regulated expression of adeno-associated virus large Rep proteins in stable 293 cell lines using the Cre/loxP switching system"J.Gen.Virol.. 80. 2477-2480 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Shintani,Y.et al.: "Induction of apoptosis after switch-on of the hepatitis B virus X gene mediated by the Cre/loxP recombination system"J.Gen.Virol.. 80. 3257-3265 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Sudo,H.et al.: "Postsynaptic expression of Ca2+-permeable AMPA-type glutamate receptor channels by viral-mediated gene transfer"Bram Ros Hol Bram Ros.. 65. 176-185 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Yamabe,K.et al.: "Cancer gene therapy using a pro-apoptotic gene,caspase-3"Gene Ther.. 6. 1952-1959 (1999)

    • Related Report
      1999 Annual Research Report

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

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