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脳機能解析に用いるノックアウトラットの開発

Research Project

Project/Area Number 15082211
Research Category

Grant-in-Aid for Scientific Research on Priority Areas

Allocation TypeSingle-year Grants
Review Section Biological Sciences
Research InstitutionNational Institute for Physiological Sciences (2004-2007)
Okazaki National Research Institutes (2003)

Principal Investigator

平林 真澄  National Institute for Physiological Sciences, 行動・代謝分子解析センター, 准教授 (20353435)

Co-Investigator(Kenkyū-buntansha) 八木 健  大阪大学, 大学院・生命機能研究科, 教授 (10241241)
保地 眞一  信州大学, 繊維学部, 准教授 (10283243)
平林 敬浩  大阪大学, 大学院・生命機能研究科, 助教 (40297015)
篠原 隆司  京都大学, 大学院・医学研究科, 教授 (30322770)
Project Period (FY) 2003 – 2007
Project Status Completed (Fiscal Year 2007)
Budget Amount *help
¥55,200,000 (Direct Cost: ¥55,200,000)
Fiscal Year 2007: ¥9,100,000 (Direct Cost: ¥9,100,000)
Fiscal Year 2006: ¥10,700,000 (Direct Cost: ¥10,700,000)
Fiscal Year 2005: ¥11,800,000 (Direct Cost: ¥11,800,000)
Fiscal Year 2004: ¥11,800,000 (Direct Cost: ¥11,800,000)
Fiscal Year 2003: ¥11,800,000 (Direct Cost: ¥11,800,000)
Keywords核移植 / クローンラット / 円形精子細胞 / 生殖幹細胞 / 遺伝子トラップ / ノックアウトラット / ROSI / 脱メチル化 / インプリンティング / トランスジェニックラット / EGFP / 精子形成 / 顕微授精 / p34^<cdc2> kinase / 精原細胞 / コンディショナルターゲッティング / Cre-loxP / dsRNAi法
Research Abstract

脳機能解析に用いるためのノックアウトラット(KO)作製を目指し、これまでに体細胞クローン法と精子幹細胞を用いた方法を検討してきた。本年度は、(1)移植体細胞核の初期化動態をゲノムの脱メチル化を指標とした解析、(2)ラット由来GS細胞に遺伝子トラップし、遺伝子破壊が確認できたクローン由来の個体作製を行った。
(1)脱メチル化を指標とした体細胞ドナー核の初期化の検討:移植体細胞核の初期化動態を調べることを目的に、DNAメチル化修飾の動態を調べた。胚発生の初期に起こる脱メチル化が初期化機構の一つであるという仮定のもと、まずラット受精卵における脱メチル化動態を調べた。交配後に受精卵を回収しメチル化量を調べたところ、受精から6〜10時間の間に急速にメチル化量が低下した。顕微授精(ICSI)で得た受精卵ではメチル化量の低下が受精卵を回収したものより僅かであった。ラットでは未受精卵を体外に取り出し操作することで、初期化機構の一つである脱メチル化能に影響を及ぼすことが明らかになった。
(2)ラット精子幹細胞を用いた遺伝子改変個体の作製:遺伝子トラップ法により遺伝子導入したラット由来生殖幹細胞(GS細胞)をG418薬剤耐性により選択後、遺伝子破壊が確認できたクローンを免疫不全マウスの精巣内に移植し、半数体精子細胞精子へと分化させた。これによりできあがった円形精子細胞をラット卵子へ顕微授精(ROSI)し、遺伝子組換えラットを作製した。SD-EGFPラット由来およびWistar-EGFPラット由来の合計2トラップクローンで、それぞれ19匹および64匹の産仔が得られ、neoをプローブとしたサザン解析の結果、15匹および16匹(含未解析14匹)にneoのバンドが検出された。ホモ個体の作製と詳細な遺伝子解析は進行中だが、ラット精子幹細胞を用いることでKOラットを作製できることが明らかになった。

Report

(5 results)
  • 2007 Annual Research Report
  • 2006 Annual Research Report
  • 2005 Annual Research Report
  • 2004 Annual Research Report
  • 2003 Annual Research Report
  • Research Products

    (27 results)

All 2008 2007 2006 2005 2004 Other

All Journal Article (17 results) (of which Peer Reviewed: 3 results) Presentation (3 results) Book (1 results) Remarks (1 results) Publications (5 results)

  • [Journal Article] Live rats resulting from injection of oocytes with spermatozoa freeze-dried and stored for one year.2008

    • Author(s)
      Hochi S
    • Journal Title

      Mol. Reprod. Dev. 75

      Pages: 890-894

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Direct comparison between ICSI-mediated DNA transfer and pronuclear DNA microinjection for producing transgenic rats.2008

    • Author(s)
      Hirabayashi M
    • Journal Title

      Exp. Anim. 57

      Pages: 145-148

    • NAID

      10024166309

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Factors affecting full-term development of rat oocytes microinjected with fresh or cryopreserved round spermatids.2008

    • Author(s)
      Hirabayashi M
    • Journal Title

      Exp. Anim. 57(印刷中)

    • NAID

      10024166601

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] No effect of recombinase-mediated DNA transfer on production efficiency of transgenic rats.2006

    • Author(s)
      Hirabayashi M.
    • Journal Title

      Exp. Anim. 55・2

      Pages: 131-135

    • NAID

      10018114716

    • Related Report
      2006 Annual Research Report
  • [Journal Article] The regulation of calcium/calmodulin-dependent protein kinase II during oocyte activation in the rat.2006

    • Author(s)
      Ito J.
    • Journal Title

      J. Reprod. Dev. 52・3

      Pages: 439-447

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Characteristics of rat round spermatids differentiated from spermatogonial cells during co-culture with Sertoli cells, assessed by flow cytometry, microinsemin ation and RT-PCR.2006

    • Author(s)
      Iwanami Y.
    • Journal Title

      Theriogenology 65・2

      Pages: 288-298

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Rats produced by interspecies spermatogonial transplantation in mice and in vitro microinsemination.2006

    • Author(s)
      Shinohara T.
    • Journal Title

      Proc. Natl. Acad. Sci. USA. 103・37

      Pages: 13624-13628

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Viable rat offspring derived from oocytes intracytoplasmically injected with freeze-dried sperm heads.2005

    • Author(s)
      Hirabayashi M.
    • Journal Title

      Zygote 13・1

      Pages: 79-85

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Contribution of high p34^<cdc2> kinase activity to premature chromosome condensation of injected somatic cell nuclei in rat oocytes.2005

    • Author(s)
      Ito J.
    • Journal Title

      Reproduction 129・2

      Pages: 171-180

    • Related Report
      2005 Annual Research Report
  • [Journal Article] No effect of recombinase-mediated DNA transfer on production efficiency of transgenic rats.2005

    • Author(s)
      Hirabayashi M.
    • Journal Title

      Exp.Anim. 55(In press)

    • NAID

      10018114716

    • Related Report
      2005 Annual Research Report
  • [Journal Article] The regulation of calcium/calmodulin-dependent protein kinase II during oocyte activation in the rat.2005

    • Author(s)
      Ito J.
    • Journal Title

      J.Reprod.Dev. 52(In press)

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Factors affecting production of transgenic rats by ICSI-mediated DNA transfer ; effects of sonication and freeze-thawing of spermatozoa, rat strains for sperm and oocyte donors, and different constructs of exogenous DNA.2005

    • Author(s)
      Hirabayashi M.
    • Journal Title

      Mol.Reprod.Dev. 70・4

      Pages: 422-428

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Viable rat offspring derived from oocytes intracytoplasmically injected with freeze-dried sperm heads.2005

    • Author(s)
      Hirabayashi M.
    • Journal Title

      Zygote (In press)

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Contribution of high p34^<cdc2> kinase activity to premature chromosome condensation of injected somatic cell nuclei in rat oocytes.2005

    • Author(s)
      Ito J.
    • Journal Title

      Reproduction (In press)

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Effect of activation regimens for rat oocytes on full-term development following round spermatid injection2004

    • Author(s)
      Kato M.
    • Journal Title

      Contem.Top.Lab.Anim.Sci. 43・2

      Pages: 13-15

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Effect of rat strains for donors and recipients on full-term development of on e-cell zygotes cultured into morulae/blastocysts2004

    • Author(s)
      Kato M.
    • Journal Title

      J.Reprod.Dev. 50・2

      Pages: 191-195

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Production of Transgenic Rats by Ooplasmic Injection of Spermatogenic Cells Exposed to Exogenous DNA : A Preliminary Study2004

    • Author(s)
      Kato M.
    • Journal Title

      Mol.Reprod.Dev. 69・2

      Pages: 153-158

    • Related Report
      2004 Annual Research Report
  • [Presentation] ラットICSI系で作製した前核期卵における染色体異常の誘発要因について.2007

    • Author(s)
      保地 眞一
    • Organizer
      第100回日本実験動物学会総会
    • Place of Presentation
      東京都文京区
    • Year and Date
      2007-10-21
    • Related Report
      2007 Annual Research Report
  • [Presentation] -196℃、+4℃、または+25℃で長期保存したフリーズドライラット精子のICSIによる個体発生能。2007

    • Author(s)
      渡辺 香
    • Organizer
      第48回日本哺乳動物卵子学会大会
    • Place of Presentation
      山梨県甲府市
    • Year and Date
      2007-05-26
    • Related Report
      2007 Annual Research Report
  • [Presentation] ラットROSIにおいて新鮮精子細胞と凍結精子細胞のどちらを用いるかで卵活性化処理の適期が異なる.2007

    • Author(s)
      加藤 めぐみ
    • Organizer
      第54回日本実験動物学会総会
    • Place of Presentation
      東京都江戸川区
    • Year and Date
      2007-05-24
    • Related Report
      2007 Annual Research Report
  • [Book] Cloning in the rat. Health Consequences of Cloning / Progress in Pharmacology and Toxicology (Eds. Inui A)2005

    • Author(s)
      Hirabayashi M.
    • Publisher
      Taylor & Francis Books, Abingdon, Oxford.
    • Related Report
      2005 Annual Research Report
  • [Remarks]

    • URL

      http://www.nips.ac.jp/mamtg/

    • Related Report
      2007 Annual Research Report
  • [Publications] Hirabayashi M.: "Factors affecting premature chromosome condensation of cumulus cell nuclei Injected into rat oocytes."J.Reprod.Dev.. 49(2). 121-126 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Hirabayashi M.: "Factors influencing chromosome condensation and development of cloned rat embryos."Cloning Stem Cells. 5(1). 35-42 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Kato M.: "Effect of activation regimens for rat oocytes on full-term development following round spermatid injection."Contem.Top.Lab.Anim.Sci.. 43(2). 10-12 (2004)

    • Related Report
      2003 Annual Research Report
  • [Publications] Kato M.: "Effect of rat strains for donors and recipients on full-term development of one-cell zygotes cultured into morulae/blastocysts."Mol.Reprod.Dev.. 50(in press). (2004)

    • Related Report
      2003 Annual Research Report
  • [Publications] Kato M.: "Production of transgenic rats by ooplasmic injection of spermatogenic cells exposed to exogenous DNA : A preliminary study."J.Reprod.Dev.. 67(in press). (2004)

    • Related Report
      2003 Annual Research Report

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Published: 2003-04-01   Modified: 2018-03-28  

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