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Development of novel method for generation of genome editing mice without ex vivo handling of embryos

Research Project

Project/Area Number 15K14371
Research Category

Grant-in-Aid for Challenging Exploratory Research

Allocation TypeMulti-year Fund
Research Field Laboratory animal science
Research InstitutionTokai University

Principal Investigator

OHTSUKA Masato  東海大学, 医学部, 准教授 (90372945)

Co-Investigator(Kenkyū-buntansha) 和田 健太  東京農業大学, 生物産業学部, 准教授 (20508113)
Co-Investigator(Renkei-kenkyūsha) SATO Masahiro  鹿児島大学, 医用ミニブタ・先端医療開発研究センター, 教授 (30287099)
Research Collaborator MIURA Hiromi  東海大学, 医学部, 特定研究員 (90599523)
Project Period (FY) 2015-04-01 – 2018-03-31
Project Status Completed (Fiscal Year 2017)
Budget Amount *help
¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000)
Fiscal Year 2017: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2016: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
Fiscal Year 2015: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Keywordsゲノム編集 / マウス / CRISPR / エレクトロポレーション / in vivo / GONAD / i-GONAD / CRISPR/Cas9 / 白内障 / piggyBac
Outline of Final Research Achievements

Animal transgenesis involves three major technical steps; isolation of fertilized eggs, microinjection and subsequent transfer of eggs into recipient females, which require sophisticated equipment as well as highly skilled personnel to perform the task. Genome-editing via Oviductal Nucleic Acids Delivery (GONAD) method developed in this study is a novel technique enabling genome editing in mice through intraoviductal instillation of genome editing components followed by an in vivo electroporation. Since the GONAD does not require ex vivo manipulation of zygotes, it is possible to produce genome-edited mice even by the researchers who do not possess special skills for ex vivo handling and micromanipulation of embryos.

Report

(4 results)
  • 2017 Annual Research Report   Final Research Report ( PDF )
  • 2016 Research-status Report
  • 2015 Research-status Report
  • Research Products

    (44 results)

All 2018 2017 2016 2015 Other

All Int'l Joint Research (4 results) Journal Article (7 results) (of which Int'l Joint Research: 4 results,  Peer Reviewed: 7 results,  Open Access: 6 results,  Acknowledgement Compliant: 1 results) Presentation (28 results) (of which Int'l Joint Research: 9 results,  Invited: 10 results) Book (1 results) Remarks (3 results) Patent(Industrial Property Rights) (1 results)

  • [Int'l Joint Research] University of Nebraska, Medical Center(米国)

    • Related Report
      2017 Annual Research Report
  • [Int'l Joint Research] Georg-August-University Gottingen(Germany)

    • Related Report
      2017 Annual Research Report
  • [Int'l Joint Research] ネブラスカ大学(米国)

    • Related Report
      2016 Research-status Report
  • [Int'l Joint Research] ネブラスカ大学医療センター(米国)

    • Related Report
      2015 Research-status Report
  • [Journal Article] Successful production of genome-edited rats by the rGONAD method2018

    • Author(s)
      Kobayashi T, Namba M, Koyano T, Fukushima M, Sato M, Ohtsuka M, Matsuyama M.
    • Journal Title

      BMC Biotechnol

      Volume: 18 Issue: 1 Pages: 19-19

    • DOI

      10.1186/s12896-018-0430-5

    • Related Report
      2017 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] i-GONAD: a robust method for in situ germline genome engineering using CRISPR nucleases2018

    • Author(s)
      Ohtsuka Masato、Sato Masahiro、Miura Hiromi、Takabayashi Shuji、Matsuyama Makoto、Koyano Takayuki、Arifin Naomi、Nakamura Shingo、Wada Kenta、Gurumurthy Channabasavaiah B.
    • Journal Title

      Genome Biology

      Volume: 19 Issue: 1 Pages: 1-15

    • DOI

      10.1186/s13059-018-1400-x

    • Related Report
      2017 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] The piggyBac-Based Gene Delivery System Can Confer Successful Production of Cloned Porcine Blastocysts with Multigene Constructs.2016

    • Author(s)
      Sato M, Maeda K, Koriyama M, Inada E, Saitoh I, Miura H, Ohtsuka M, Nakamura S, Sakurai T, Watanabe S, Miyoshi K.
    • Journal Title

      Int J Mol Sci.

      Volume: 17 Issue: 9 Pages: 1424-1424

    • DOI

      10.3390/ijms17091424

    • NAID

      130007023583

    • Related Report
      2016 Research-status Report
    • Peer Reviewed / Open Access
  • [Journal Article] Nucleic acids delivery methods for genome editing in zygotes and embryos: The old, the new, and the old-new.2016

    • Author(s)
      Sato M, Ohtsuka M, Watanabe W, Gurumurthy CB
    • Journal Title

      Biology Direct

      Volume: 11 Issue: 1 Pages: 16-16

    • DOI

      10.1186/s13062-016-0115-8

    • Related Report
      2016 Research-status Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] GONAD: a novel CRISPR/Cas9 genome editing method that does not require ex vivo handling of embryos2016

    • Author(s)
      Gurumurthy C.B., Takahashi G., Wada K., Miura H., Sato M., and Ohtsuka M.
    • Journal Title

      Curr. Protoc. Hum. Genet.

      Volume: 88 Issue: 1

    • DOI

      10.1002/0471142905.hg1508s88

    • Related Report
      2015 Research-status Report
    • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
  • [Journal Article] GONAD: Genome-editing via Oviductal Nucleic Acids Delivery system: a novel microinjection independent genome engineering method in mice.2015

    • Author(s)
      Takahashi G, Gurumurthy CB, Wada K, Miura H, Sato M, Ohtsuka M
    • Journal Title

      Scientific Reports

      Volume: 5 Issue: 1 Pages: 11406-11406

    • DOI

      10.1038/srep11406

    • Related Report
      2015 Research-status Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] PiggyBac transposon-mediated gene delivery efficiently generates stable transfectants derived from cultured primary human deciduous tooth dental pulp cells (HDDPCs) and HDDPC-derived iPS cells.2015

    • Author(s)
      Inada E, Saito I, Watanabe S, Aoki R, Miura H, Ohtsuka M, Murakami T, Sawami T, Yamasaki Y, Sato M
    • Journal Title

      International Journal of Oral Science

      Volume: 7 Issue: 3 Pages: 144-145

    • DOI

      10.1038/ijos.2015.18

    • Related Report
      2015 Research-status Report
    • Peer Reviewed / Open Access
  • [Presentation] ex vivo胚操作が不要なゲノム編集動物作製法「GONAD」の開発とラットへの応用を目指して2018

    • Author(s)
      大塚正人
    • Organizer
      第11回ラットリソースリサーチ研究会
    • Related Report
      2017 Annual Research Report
    • Invited
  • [Presentation] i-GONAD: A Method for Genome Editing Rodents without ex vivo Handling of Embryos2018

    • Author(s)
      Ohtsuka M
    • Organizer
      New Frontiers in CRISPR-based Gene Editing (Part of the 25th International Molecular Medicine Tri-Conference)
    • Related Report
      2017 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] 採卵、顕微注入、胚移植のステップが不要なゲノム編集マウス作製法GONADによるノックインマウス作製2017

    • Author(s)
      大塚 正人, 三浦 浩美, 佐藤 正宏
    • Organizer
      日本ゲノム編集学会第2回大会
    • Related Report
      2017 Annual Research Report
  • [Presentation] Rescue of the albino phenotype in mice using GONAD method2017

    • Author(s)
      Hiromi Miura, Naomi Arifin, Masahiro Sato, Masato Ohtsuka
    • Organizer
      International Pigment Cell Conference 2017(IPCC2017)
    • Related Report
      2017 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Development of a mouse model suitable for in vivo genome editing efficiency studies2017

    • Author(s)
      Hiromi Miura, Yutaka Inagaki,Channabasavaiah B Gurumurthy, Masahiro Sato, Masato Ohtsuka
    • Organizer
      14th Transgenic Technology Meeting (TT2017)
    • Related Report
      2017 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Improved GONAD (i-GONAD) (I) as ex vivo manipulation-free genome-editing system allowing efficient knock-out, large deletion, and knock-in2017

    • Author(s)
      Masato Ohtsuka, Hiromi Miura, Channabasavaiah B. Gurumurthy, Masahiro Sato
    • Organizer
      14th Transgenic Technology Meeting (TT2017)
    • Related Report
      2017 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Improved GONAD (i-GONAD) (II): usefulness of rhodamine-dextran for monitoring the success of the GONAD and of gonadotrophin-based regulation of the timing for the GONAD2017

    • Author(s)
      Masahiro Sato, Shingo Nakamura, Channabasavaiah B. Gurumurthy, Satoshi Watanabe, Masato Ohtsuka
    • Organizer
      14th Transgenic Technology Meeting (TT2017)
    • Related Report
      2017 Annual Research Report
    • Int'l Joint Research
  • [Presentation] 新たなゲノム編集技術GONAD法を用いた遺伝子改変ラットの作製法2017

    • Author(s)
      小林 朋絵、難波 真澄、古家野 孝行、佐藤 正宏、大塚 正人、松山 誠
    • Organizer
      第40回日本分子生物学会年会
    • Related Report
      2017 Annual Research Report
  • [Presentation] 簡便なゲノム編集マウス作製法GONAD法を用いたアルビノマウスのレスキュー実験2017

    • Author(s)
      高林 秀次、青島 拓也、佐藤 正宏、大塚 正人
    • Organizer
      第40回日本分子生物学会年会
    • Related Report
      2017 Annual Research Report
  • [Presentation] In vivoゲノム編集効率評価系モデルマウスの開発2017

    • Author(s)
      三浦 浩美、稲垣 豊、佐藤 正宏、大塚 正人
    • Organizer
      第40回日本分子生物学会年会
    • Related Report
      2017 Annual Research Report
  • [Presentation] GONAD: a simple method to generate genome-edited mice without ex vivo handling of embryos. Mouse genome engineering2017

    • Author(s)
      Ohtsuka M
    • Organizer
      Mouse genome engineering in EMBO Practical Course
    • Related Report
      2017 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] 遺伝子改変マウス作製の簡便化を実現する新しい手法: GONAD(Genome-editing via Oviductal Nucleic Acids Delivery)法2017

    • Author(s)
      大塚正人
    • Organizer
      徳島大学「ゲノム編集事業」キックオフシンポジウム
    • Related Report
      2017 Annual Research Report
    • Invited
  • [Presentation] 新規遺伝子改変マウス作製方法:PITT, Easi-CRISPR, GONAD2017

    • Author(s)
      大塚正人
    • Organizer
      第3回川島カンファレンス
    • Related Report
      2017 Annual Research Report
    • Invited
  • [Presentation] GONAD: ex vivo胚操作を要しないゲノム編集マウス作製手法2017

    • Author(s)
      大塚正人
    • Organizer
      第40回日本分子生物学会年会 ランチョンセミナー
    • Related Report
      2017 Annual Research Report
    • Invited
  • [Presentation] GONAD法:採卵、顕微注入、胚移植のステップが不要なゲノム編集マウス作製法2016

    • Author(s)
      大塚 正人、高橋 剛、三浦 浩美、和田 健太、佐藤 正宏
    • Organizer
      第39回日本分子生物学会年会
    • Place of Presentation
      パシフィコ横浜(神奈川県横浜市)
    • Year and Date
      2016-11-30
    • Related Report
      2016 Research-status Report
    • Invited
  • [Presentation] 医用ブタおける遺伝子組換え動物作製の実際とその可能性について2016

    • Author(s)
      佐藤 正宏、渡部 聡、大塚 正人
    • Organizer
      第50回日本実験動物技術者協会総会
    • Place of Presentation
      ウェスタ川越(埼玉県川越市)
    • Year and Date
      2016-09-29
    • Related Report
      2016 Research-status Report
    • Invited
  • [Presentation] ex vivo胚操作が不要なゲノム編集マウス作製法2016

    • Author(s)
      大塚 正人, 高橋 剛, 和田 健太, 三浦 浩美, 佐藤 正宏
    • Organizer
      平成28年度新学術領域研究「学術研究支援基盤形成【先端モデル動物支援プラットフォーム】」若手支援技術講習会
    • Place of Presentation
      蓼科グランドホテル滝の湯(長野県茅野市)
    • Year and Date
      2016-09-14
    • Related Report
      2016 Research-status Report
  • [Presentation] 採卵、顕微注入、胚移植を要しないゲノム編集マウス作製法GONADによるノックアウトマウス作製2016

    • Author(s)
      大塚 正人, 高橋 剛, 和田 健太, 三浦 浩美, Channabasavaiah B Gurumurthy, 佐藤 正宏
    • Organizer
      第63回日本実験動物学会総会
    • Place of Presentation
      ミューザ川崎シンフォニーホール(神奈川県川崎市)
    • Year and Date
      2016-05-18
    • Related Report
      2016 Research-status Report
  • [Presentation] Improvement of GONAD (genome-editing via oviductal nucleic acids delivery), one of the zygote injection-free genome editing systems targeted to preimplantation embryos2016

    • Author(s)
      Masahiro Sato, Channabasavaiah B. Gurumurthy, Satoshi Watanabe, Masato Ohtsuka
    • Organizer
      13th Transgenic Technology Meeting (TT2016)
    • Place of Presentation
      Prague, Czech Republic
    • Year and Date
      2016-03-20
    • Related Report
      2015 Research-status Report
    • Int'l Joint Research
  • [Presentation] GONAD and Easy (Isi)-CRISPR: novel mouse genome engineering tools2016

    • Author(s)
      Masato Ohtsuka
    • Organizer
      13th Transgenic Technology Meeting (TT2016)
    • Place of Presentation
      Prague, Czech Republic
    • Year and Date
      2016-03-20
    • Related Report
      2015 Research-status Report
    • Int'l Joint Research / Invited
  • [Presentation] GONAD法:ex vivo 胚操作を要しないゲノム編集動物作製法2016

    • Author(s)
      大塚正人
    • Organizer
      SKIP×幹細胞若手の会 Symposium 2016 ~Integrative Stem Cell Biology for Regenerative Medicine~
    • Place of Presentation
      御茶ノ水トライエッジカンファレンス(東京都千代田区)
    • Related Report
      2016 Research-status Report
    • Invited
  • [Presentation] zygote injectionに依らない生殖細胞、胚を標的とした遺伝子導入によるCRISPR/Cas9 genome editingの可能性2015

    • Author(s)
      佐藤正宏、大塚正人、中村伸吾、桜井敬之、稲田絵美、齋藤一誠、渡部聡
    • Organizer
      第38回日本分子生物学会年会(BMB2015)
    • Place of Presentation
      神戸ポートアイランド (兵庫県神戸市)
    • Year and Date
      2015-12-01
    • Related Report
      2015 Research-status Report
  • [Presentation] GONAD: a novel CRISPR/Cas9 genome editing method that does not require ex vivo handling of fertilized eggs2015

    • Author(s)
      Masato Ohtsuka, Gou Takahashi, Kenta Wada, Hiromi Miura, Channabasavaiah B Gurumurthy, Masahiro Sato
    • Organizer
      29th International Mammalian Genome Conference (IMGC)
    • Place of Presentation
      Yokohama Port Opening Memorial Hall, (Yokohama, Kanagawa, Japan)
    • Year and Date
      2015-11-08
    • Related Report
      2015 Research-status Report
    • Int'l Joint Research
  • [Presentation] GONAD:採卵、顕微注入、移植を要しない新規ゲノム編集マウス作製法2015

    • Author(s)
      大塚正人、Channabasavaiah B Gurumurthy、三浦浩美、佐藤正宏、和田健太、高橋剛
    • Organizer
      第108回日本繁殖生物学会大会
    • Place of Presentation
      宮崎大学 木花キャンパス (宮崎県宮崎市)
    • Year and Date
      2015-09-17
    • Related Report
      2015 Research-status Report
  • [Presentation] GONAD法:卵管を介したマウス2細胞期胚へのin vivo RNA導入による新規ゲノム編集マウス作製法2015

    • Author(s)
      大塚正人、佐藤正宏、高橋剛
    • Organizer
      GE Life Sciences Day 2015
    • Place of Presentation
      パシフィコ横浜会議センター (神奈川県横浜市)
    • Year and Date
      2015-07-24
    • Related Report
      2015 Research-status Report
  • [Presentation] ex vivo 処置を介さない CRISPR/Cas9 系による遺伝子改変マウス作製法「GONAD」 の開発2015

    • Author(s)
      高橋 剛、Channabasavaiah B Gurumurthy、和田 健太、三浦 浩美、佐藤 正 宏、大塚 正人
    • Organizer
      第 12 回北海道実験動物研究会総会・学術集会
    • Place of Presentation
      北海道大学大学院医学研究科臨床講義棟第三講堂 (北海道札幌市)
    • Year and Date
      2015-07-11
    • Related Report
      2015 Research-status Report
  • [Presentation] 採卵、顕微注入、胚移植を要しない新規ゲノム編集マウス作製法2015

    • Author(s)
      高橋剛、和田健太、三浦浩美、佐藤正宏、大塚正人
    • Organizer
      第 29 回モロシヌス研究会
    • Place of Presentation
      神戸 かんぽの宿 有馬 (兵庫県神戸市)
    • Year and Date
      2015-07-03
    • Related Report
      2015 Research-status Report
  • [Presentation] 卵管を介したマウス 2 細胞期胚への RNA 導入法の開 発とゲノム編集技術への応用2015

    • Author(s)
      高橋剛、和田健太、三浦浩美、佐藤正宏、大塚正人
    • Organizer
      第62回日本実験動物学会総会
    • Place of Presentation
      京都テルサ (京都府京都市)
    • Year and Date
      2015-05-28
    • Related Report
      2015 Research-status Report
  • [Book] CRISPR/Cas9 and the Paradigm Shift in Mouse Genome Manipulation Technologies. In: Turksen K (ed) Genome editing2016

    • Author(s)
      Gurumurthy C.B., Quadros R., Sato M., Mashimo T., Lloyd K.C.K. and Ohtsuka M
    • Total Pages
      13
    • Publisher
      Springer International Publishing
    • Related Report
      2016 Research-status Report
  • [Remarks]

    • URL

      http://masato2.wixsite.com/ohtsuka-lab

    • Related Report
      2017 Annual Research Report
  • [Remarks] 東海大学医学部基礎医学系分子生命科学 大塚研究室

    • URL

      http://masato2.wixsite.com/ohtsuka-lab

    • Related Report
      2016 Research-status Report
  • [Remarks]

    • URL

      http://masato2.wix.com/ohtsuka-lab

    • Related Report
      2015 Research-status Report
  • [Patent(Industrial Property Rights)] 哺乳動物の受精卵に物質を導入する方法2017

    • Inventor(s)
      大塚 正人、森泉 俊幸、森泉 康裕
    • Industrial Property Rights Holder
      株式会社ベックス、学校法人東海大学
    • Industrial Property Rights Type
      特許
    • Filing Date
      2017
    • Related Report
      2017 Annual Research Report

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Published: 2015-04-16   Modified: 2022-06-07  

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