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Novel expression cloning methods based on retrovirus mediated gene transfer.

Research Project

Project/Area Number 10559006
Research Category

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

Allocation TypeSingle-year Grants
Section展開研究
Research Field 広領域
Research InstitutionThe University of Tokyo

Principal Investigator

KITAMURA Toshio  Institute of Medical Science, The University of Tokyo Visiting Professor, 医科学研究所, 客員教授 (20282527)

Co-Investigator(Kenkyū-buntansha) NOSAKA Tetsuya  Institute of Medical Science, The University of Tokyo Visiting Associate Professor, 医科学研究所, 客員助教授 (30218309)
Project Period (FY) 1998 – 2000
Project Status Completed (Fiscal Year 2000)
Budget Amount *help
¥12,900,000 (Direct Cost: ¥12,900,000)
Fiscal Year 2000: ¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 1999: ¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 1998: ¥6,600,000 (Direct Cost: ¥6,600,000)
Keywordsretrovirus / expression cloning / cDNA library / signal sequence trap / GFP / サイトカイン / サイトカインレセプター / TNFレセプター / 転写因子
Research Abstract

We previously established a high-efficiency retrovirus-mediated expression cloning method. Using this system, we have now developed a novel expression cloning method in which cDNAs can be isolated based on the subcellular localization of their products. We make cDNAs using random hexamer and fuse them to 5' endo of the cDNA of green fluorescent protein(GFP)in the pMX retrovirus vector to construct a cDNA-GFP fusion retrovirus library. The derived retroviruses are then infected to NIH3T3 cells to identify a cDNA of interest based on the subcellular localization of its protein product such as nucleus, nucleoli, Golgi apparatus, and cell surface. Using this strategy, we have identified a novel zinc finger protein from fetal mouse liver cells that contain hematopoietic progenitor cells.
We have also established a novel signal sequence trap method which can identify cDNA fragments containing signal sequence that encode secreted and cell-surface. Signal sequence trap was originally developed in Kyoto University, and was improved by a group in Genentech, isolate cDNAs with signal sequences that encode such proteins have been established. In our method termed, cDNA fragments fused to an extracellular deletion mutant of the constitutively active receptor for thrombopoietin(MPL)were introduced into IL-3-dependent cells via retrovirus infection followed by the selection of factor-independent clones. Our method is much quicker and more accurate than the previously published methods. We have identified three novel receptors(a type I cytokine receptor, a TNF receptor-like molecule, and a novel low-density lipoprotein receptor-related protein)and several novel secreted molecules by SST-REX, and are now characterizing these molecules using various strategies including knockout mice.

Report

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

    (50 results)

All Other

All Publications (50 results)

  • [Publications] Fujio, K.: "Molecular cloning of a novel type I cytokine receptor similar to the common gamma chain."Blood. 95. 2204-2211 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Misawa, K.: "Molecular cloning and tissue distrubution of a mouse homologue of human LIGHT, a member of TNF super family."Cytogenetics and Cell Genetics. 89. 89-91 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Misawa, K.: "A novel method to identify cDNAs based on localization of the GFP-fusion products."Proc.Natl.Acad.Sci.U.S.A.. 97. 3062-3066 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Morita, S.: "An efficient and stable retrovirus packaging system for transient packaging."Gene Therapy. 7. 1063-1066 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Tsuruga, H.: "Identification of novel membrane and secreted proteins upregulated during adipocyte differentiation."Biochem.Biophys.Res.Commun.. 272. 293-297 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Kojima, T.: "Troy, a newly identified member of the tumor necrosis factor receptor superfamily exhibits a homology with Edar and is expressed in embryonic skin and hair follicles."J.Biol.Chem.. 275. 20742-20747 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Kawashima, T.: "MgcRacGAP is involved in the control of growth and differentiation of hematopoietic cells."Blood. 96. 2116-2124 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Sugiyama, T.: "A novel low-density lipoprotein receptor-related protein mediating cellular uptake of apolipoprotein E-enriched β-VLDL in vitro."Biochemistry. 39. 15817-15825 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Hirose, K.: "Involvement of MgcRacGAP in cytokinesis through associating with mitotic spindle and midbody."J.Biol.Chem.. (in press). (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Fujio, K., Nosaka, T., Kojima, T., Kawashima, T., Yahata, T., Copeland, N.G., Gilbert, D.J., Jenkins, N.A., Yamamoto, K, Nishimura, T., and Kitamura, T.: "Molecular cloning of a novel type I cytokine receptor similar to the common gamma chain."Blood. 95. 2204-2211 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Misawa, K., Nosaka, T., Kojima, T., Hirai, M.and Kitamura, T.: "Molecular cloning and characterization of a mouse homolog of human TNFSF14, a member of the TNF superfamily."Cytogenetics and Cell Genetics. 89. 89-91 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Misawa, K., Morita, S., Nosaka, T., Kaneko, A., Nakahata, T., Asano, S.and Kitamura, T.: "A method to identify cDNAs based on localization of green fluorescent protein fusion products."Proc.Natl.Acad.Sci.U.S.A.. 97. 3062-3066 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Morita, S., Kojima, T., and Kitamura, T.: "Plat-E : an efficient and stable system for transient packaging of retroviruses."Gene Therapy. 7. 1063-1066 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Tsuruga, H., Kumagai, H., Kojima, T., and Kitamura, T.: "Identification of novel membrane and secreted proteins upregulated during adipocyte differentiation."Biochem.Biophys.Res.Commun.. 272. 293-297 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Kojima, T., Morikawa, Y., Copeland, N.G., Gilbert, D.J., Jenkins, N.A., Senba, E.and Kitamura, T.: "Troy, a newly identified member of the tumor necrosis factor receptor superfamily, exhibits a homology with Edar and is expressed in embryonic skin and hair follicles."J.Biol.Chem.. 275. 20742-20747 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Kawashima, T., Hirose, K., Satoh, T., Ikeda, Y., Kaziro, Y., Nosaka, T.and Kitamura, T.: "MgcRacGAP is involved in the control of growth and differentiation of hematopoietic cells."Blood. 96. 2116-2124 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Sugiyama, T., Kumagai, H., Morikawa, Y., Wada, Y., Sugiyama, A., Yasuda, K., Yokoi, N., Kojima, T., Nosaka, T., Senba, E., Kimura, S., Kadowaki, T., Kodama, T., and Kitamura, T.: "A novel low-density lipoprotein receptor-related protein mediating cellular uptake of apolipoprotein E-enriched β-VLDL in vitro."Biochemistry. 39. 15817-15825 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Hirose, K., Kawashima, T., Iwamoto, I., Nosaka, T., and Kitamura, T.: "Involvement of MgcRacGAP in cytokinesis through associating with mitonic spindle and midbody."J.Biol.Chem.. (in press). (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Fujio,K.: "Molecular cloning of a novel type I cytokine receptor similar to the common gamma chain."Blood. 95. 2204-2211 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Misawa,K.: "Molecular cloning and tissuedistrubution of a mouse homologue of human LIGHT, a member of TNF super family."Cytogenetics and Cell Genetics. 89. 89-91 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Misawa,K.: "A novel method to identify cDNAs based on localization of the GFP-fusion products."Proc.Natl.Acad.Sci.U.S.A.. 97. 3062-3066 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Morita,S.: "An efficient and stable retrovirus packaging system for transient packaging."Gene Therapy. 7. 1063-1066 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Tsuruga,H.: "Identification of novel membrane and secreted proteins upregulated during adipocyte differentiation."Biochem.Biophys.Res.Commun.. 272. 293-297 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Kojima,T.: "Troy, a newly identified member of the tumor necrosis factor receptor superfamily exhibits a homology with Edar and is expressed in embryonic skin and hair follicles."J.Biol.Chem.. 275. 20742-20747 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Kawashima,T.: "MgcRacGAP is involved in the control of growth and differentiation of hematopoietic cells."Blood. 96. 2116-2124 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Sugiyama,T.: "A novel low-density lipoprotein receptor-related protein mediating cellular uptake of apolipoprotein E-enriched β-VLDL in vitro."Biochemistry. 39. 15817-15825 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Hirose,K.: "Involvement of MgcRacGAP in cytokinesis through associating with mitotic spindle and midbody."J.Biol.Chem.. (in press). (2001)

    • Related Report
      2000 Annual Research Report
  • [Publications] Yang,Y-L.: "Receptors for polytropic and xenotropic murine leukemia viruses encoded by a single gane at Rmc1"Nature Genetics. 21. 216-219 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Nosaka,T.: "Identification and characterizarion of constitutively active STAT5"Molecular Biology of Hematopoiesis 6. 277-287 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Kojima,T.: "A novel signal sequence trap method using retrovirus-mediated gene transfer"Nature Biotechnol.. 17. 487-490 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Nosaka,T.: "STAT5 as a molecular regulator of proliferation,differentiation,and apoptosis in hematopoietic cells"EMBO J.. 18. 4754-4765 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Kitamura,T.: "Isolation of cDNAs for T cell antigens by retrovirs-mediated expression cloning"T Cell Protocols,Methods in Molecular Biology.. 143-152 (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] Fujio,K.: "Molecular cloning of a novel type I cytokine receptor"Blood. (in press). (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] Misawa,K.: "Molecular cloning and tissue distrubution of a mouse homologue of human LIGHT,a member of TNF super family"Cytogenetics and Cell Genetics. (in press). (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] Misawa,K.: "A novel method to identify cDNAs based on localization of the GFP-fusion products"Proc.Natl.Acad.Sci.U.S.A.. (in press). (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] Morita,S: "An efficient and stable retrovirus packaging system for transient packaging"Gene Therapy. (in press). (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] Onishi,M.: "Cytokine receptors:Structures and signal transduction." Int.Rev.Immunol.16. 617-634 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Kitamura,T.: "New experimental approaches in retrovirus-mediated expression screeing." Int.J.Hematol.67. 351-359 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Hata,D.: "Involvement of Bruton's tyrosine kinase in FceRI-dependent mast cell degranulation and cytokine production." J.Exp.Med.187. 1235-1247 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Morikawa,Y.: "induction of synaptosomal-associated protein-23kd(SNAP-23)by various cytokines." Blood. 92. 129-135 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Onishi,M.: "Identification and characterization of a constitutive active STAT5 mutant that promotes cell proliferation." Mol.Cell.Biol.18. 3871-3879 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Jinno,A.: "Identification of the chemokine receptor TER1/CCR8 expressed in brain-derived cells and T-cells as a new coreceptor for HIV-1 infection." Biophys.Biochem.Res.Commun.243. 497-502 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Kawashima,T.: "Interleukin-12 induces tyrosine phosphorylation of an 85-kDa protein associated with the interleukin-12 receptor β1 subunit." Cell.Immunol.186. 39-44 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Kitamura,T.: "Activating mutations of the transmembrane domain of MPL in vitro and in vivo: Incorrect sequence of MPL-K,an alternative spliced form of MPL." Blood. 92. 2596-2597 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Dairaghi,D.J.: "RANTES-induced activation correlates with CD3 expression." J.Immunol.168. 426-433 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Duan,Y.: "Expression and functional analysis of a hemopoietic progenitor antigen,NJ-1 (114/A10),in a megakaryocytic lineage." Biochem.Biopys.Res.Commun.253. 401-406 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Kitamura,T.: "Isolation of cDNAs for T cell antigens by retrovirs-mediated expression cloning." Methods in Molecular Biology.(in press). (1999)

    • Related Report
      1998 Annual Research Report
  • [Publications] Nosaka,T.: "Identification and characterization of constitutively active STAT5." Molecular Biology of Hematopoiesis.(in press). (1999)

    • Related Report
      1998 Annual Research Report
  • [Publications] Mukasa,R.: "Core 2-containing O-glycans on CD43 are preferentially expressed in the memory subset of human CD4 T cells." Int.Immunol.(in press). (1999)

    • Related Report
      1998 Annual Research Report
  • [Publications] Matsumura,I.: "Involvement of cyclin D1 in STAT5-mediated cell growth." EMBO J. (in press). (1999)

    • Related Report
      1998 Annual Research Report

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

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