• Search Research Projects
  • Search Researchers
  • How to Use
  1. Back to project page

2000 Fiscal Year Final Research Report Summary

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
Keywordsretrovirus / expression cloning / cDNA library / signal sequence trap / GFP
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.

  • Research Products

    (18 results)

All Other

All Publications (18 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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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

URL: 

Published: 2002-03-26  

Information User Guide FAQ News Terms of Use Attribution of KAKENHI

Powered by NII kakenhi