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Development of efficient micro-scale gene transfer technique by combination of retrovirus vector and electroporation.

Research Project

Project/Area Number 11557131
Research Category

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

Allocation TypeSingle-year Grants
Section展開研究
Research Field Morphological basic dentistry
Research InstitutionOsaka University

Principal Investigator

KURISU Kojiro  Graduate School of Dentistry, Osaka University, Professor, 大学院・歯学研究科, 教授 (50028346)

Co-Investigator(Kenkyū-buntansha) HIGUCHI Yoshinobu  Chugai Pharmaceutical Co., LTD., Researcher, 研究員
KATO Jyoji  Graduate School of Dentistry, Osaka University, Research Associate, 大学院・歯学研究科, 助手 (90243245)
IWAMOTO Masahiro  Graduate School of Dentistry, Osaka University, Assistant Professor, 大学院・歯学研究科, 講師 (30223431)
Project Period (FY) 1999 – 2000
Project Status Completed (Fiscal Year 2000)
Budget Amount *help
¥8,200,000 (Direct Cost: ¥8,200,000)
Fiscal Year 2000: ¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 1999: ¥5,700,000 (Direct Cost: ¥5,700,000)
Keywordsmaxillofacial / gene / transfection / mouse / morphogenesis / development / organ culture / expression vector
Research Abstract

The Goal of present study is to develop efficient and reversible microscale gene delivery technique as a tool for an analysis of molecular regulation of oro-facial tissue morphogenesis. For this porpose we carried out three lines of experiments. (1) Retroviral gene transfer is known as an efficient gene transfer technique. Cre-lox-P system is a way of controlled expression of introduced gene. Thus we first tried to combine these advantages in a single vector system. Lox-P sequences were introduced in 5 and 3 prime of the cloning site of an avian retrovirus vector RCAS(E). We then subcloned Green fluorescent protein gene(GFP) into the above vector (RCAS(E)-GFP). Cre recombinase was subcloned into RCAS(B) vector (RCAS(B)-Cre). More than 70% of RCAS(E)-GFP infected fibroblast were GFP positive within 1 week after infection. We then superinfected RCAS(B)-Cre into the same cells to turn off the expression of GFP.After 3 days, most of the doubleinfected cells became GFP negative. Thus these vectors turned out to be effective for efficient and controllable gene transfer. (2) Preparation of virus particle is cumbersome and needs a lot of experience. Therefore in the second experiment, we applied electroporation for microscale gene delivery of vector cDNAs instead of virus infection. We tested various conditions for electroporation and found that the pulses for 4 times at 20-60 V/cm for 50 msec was the most efficient. We also designed micro electrode for microscale gene delivery. (3) In the last experiment, we tested whether above technique is feasible to introduce foreign gene into developing mouse tooth germs. We injected Sonic hedgehog gene(Shh) into enamel knot and applied electroporation. We fixed the explants two days later and confirmed the expression of Shh specifically in enamel knot.

Report

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

    (35 results)

All Other

All Publications (35 results)

  • [Publications] M.Iwamoto, et al.: "Transcription factor ERG variants and functional diversification of chondrocytes during limb long bone development."J.Cell Biol.. 150. 27-40 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] J.G.Liu, et al.: "Parathyroid hormone-related peptide is involved in protection against invasion of tooth germs by bone via promoting the differentiation of osteoclasts during tooth development."Mech.Dev.. 95. 189-200 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] T.Ohnishi, et al.: "Altered expression level of calbindin D28k in the periodontal ligament of rat molar in response to changes in occlusal force."J.Periodont.Res.. 35. 301-309 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] M.Enomoto-Iwamoto et al.: "Hedgehog proteins stimulate chondrogenic cell differentiation and cartilage formation."J.Bone Miner.Res.. 15. 1659-1668 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] S.Soma, et al.: "Local and chronic application of PTH accelerates tooth movement in rats."J.Dent.Res.. 79. 1717-1734 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Y.Atsumi, et al.: "Immunohistochemical detection of heme oxygenase-2 in the periodontal Ruffini ending of the rat incisor."Brain Res.. 855. 287-291 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] T.Maeda, et al.: "Molecular cloning and characterization of a novel gene, CORS26, encoding a putative secretory protein and its possible involvement in skeletal development."J Biol Chem.. 276. 3628-3634 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] 岩本容泰 他: "歯科インプラン"先端医療技術研究書. 447 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Enomoto-Iwamoto, M.et al.: "Bone morphogenetic protein signaling is required for maintenance of differentiated phenotype, control of proliferation, and hypertrophy in chondrocytes."J.Cell Biol.. 140. 409-418 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Wakisaka, S.et al.: "Immunohistochemical observation of growth associated protein-43 (GAP-43) in the developing circumvallate papilla of the rat."Cell Tissue Res.. 293. 499-507 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Matsumura, T.et al.: "Ameloblast-lineage cells of rat tooth germs proliferate and scatter in response to hepatocyte growth factor in culture."Int.J.Dev.Biol.. 42. 1137-1142 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Soma, S.et al.: "Effects of continuous infusion of PTH on experimental tooth movement in rats."J.Bone Miner.Res.. 14. 546-554 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Atsumi, Y.et al.: "Immunohistochemical localization of S-100 in the dental pulp of the rat molar."

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Youn, S.H.et al.: "Alteration in the expression level of calbindin D28k in the periodontal ligament of rat molar during experimental tooth movement of rat molar."Brain Res.. 818. 515-519 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Iwamoto, M.et al.: "Actions of hedgehog proteins on skeletal cells."Crit.Rev.Oral Biol.Med.. 10. 477-486 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Iwamoto, M.et al.: "Transcription factor ERG variants and functional diversification of chondrocytes during limb long bone development."J.Cell Biol.. 150. 27-40 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Atsumi, Y.et al.: "Effects of neonatal injury of the inferior alveolar nerve on the development and regeneration of periodontal nerve fibers in the rat incisor."Brain Res.. 871. 201-209 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Liu, J-G.et al.: "Parathyroid hormone-related peptide is involved in protection against invasion of tooth germs by bone via promoting the differentiation of osteoclasts during tooth development."Mech.Dev.. 95. 189-200 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Enomoto-Iwamoto, M.et al.: "Hedgehog proteins stimulate chondrogenic cell differentiation and cartilage formation."J.Bone Miner.Res.. 15. 1659-1668 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Soma, S.et al.: "Local and chronic application of PTH accelerates tooth movement in rats."J.Dent.Res.. 79. 1717-1734 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] M.Iwamoto, et al.: "Transcription factor ERG variants and functional diversification of chondrocytes during limb long bone development."J.Cell Biol.. 150. 27-40 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] J.G.Liu, et al.: "Parathyroid hormone-related peptide is involved in protection against invasion of tooth germs by bone via promoting the differentiation of osteoclasts during tooth development."Mech.Dev.. 95. 189-200 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] T.Ohnishi, et al.: "Altered expression level of calbindin D28k in the periodontal ligament of rat molar in response to changes in occlusal force."J.Periodont.Res.. 35. 301-309 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] M.Enomoto-Iwamoto et al.: "Hedgehog proteins stimulate chondrogenic cell differentiation and cartilage formation."J.Bone Miner.Res.. 15. 1659-1668 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] S.Soma, et al.: "Local and chronic application of PTH accelerates tooth movement in rats."J.Dent.Res.. 79. 1717-1734 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Y.Atsumi, et al.: "Immunohistochemical detection of heme oxygenase-2 in the periodontal Ruffini ending of the rat incisor."Brain Res.. 855. 287-291 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] T.Maeda, et al.: "Molecular cloning and characterization of a novel gene, CORS26, encoding a putative secretory protein and its possible involvement in skeletal development."J Biol Chem.. 276. 3628-3634 (2001)

    • Related Report
      2000 Annual Research Report
  • [Publications] 岩本容泰 他: "歯科インプラント"先端医療技術研究書. 447 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] 田端 純 他: "派の発生に関与する遺伝子とその役割"日本咀嚼誌. 9(印刷中). (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] 田端 純 他: "アンチセンス法と器官培養法による歯胚発生の研究へのアプローチ"ACBTE. 6. 19-33 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] K. Yamagi et al.: "Matrix GLA protein is a developmental regulator of chondrocyte mineralization and, when constitutively expressed, blocks endochondoral and intramembranous ossification in the limb"J. Cell Biol.. 147. 1097-1108 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] S. Soma et al.: "Effects of continuous infusion of PTH on experimental tooth movement in rats"J. Bone Mineral Res.. 14. 546-554 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] M. Iwamoto et al.: "Action of hedgehog protein on skeletal cells"Crit. Rev. Oral Biol. Med.. 10. 477-486 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Y. Atsumi et al.: "Altered distribution of Schwann cells in the periodontal ligament of the rat incisor following resection of the inferior alveolar nerve: a immunohistochemical tudy on S-100 proteins."Brain Res.. 849. 187-195 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] 岩本容泰 他: "歯科インプラント(インプラント体周囲の硬組織の再生)"先端医療技術研究所. 447 (2000)

    • Related Report
      1999 Annual Research Report

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

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