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The proposal of novel experimental system using original knockdown methods in mice

Research Project

Project/Area Number 16H04685
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
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) 中村 伸吾  防衛医科大学校(医学教育部医学科進学課程及び専門課程、動物実験施設、共同利用研究施設、病院並びに防衛, 防衛医学研究センター 医療工学研究部門, 講師 (00505323)
幸谷 愛  東海大学, 医学部, 教授 (00517477)
角田 茂  東京大学, 大学院農学生命科学研究科(農学部), 准教授 (80345032)
Research Collaborator MIURA Hiromi  
Project Period (FY) 2016-04-01 – 2019-03-31
Project Status Completed (Fiscal Year 2018)
Budget Amount *help
¥17,680,000 (Direct Cost: ¥13,600,000、Indirect Cost: ¥4,080,000)
Fiscal Year 2018: ¥5,460,000 (Direct Cost: ¥4,200,000、Indirect Cost: ¥1,260,000)
Fiscal Year 2017: ¥5,720,000 (Direct Cost: ¥4,400,000、Indirect Cost: ¥1,320,000)
Fiscal Year 2016: ¥6,500,000 (Direct Cost: ¥5,000,000、Indirect Cost: ¥1,500,000)
Keywordsノックダウン / miRNA / 遺伝子治療 / トランスジェニック / マウスモデル / 発生工学
Outline of Final Research Achievements

The purpose of this study is to establish novel strategies useful for in vivo analyses of gene function and for future gene therapies. We utilized original knockdown methods to create mouse models for 1) conditional gene recovery system, 2) conditional in vivo expression of endogenous miRNA, and 3) conditional gene knockdown by insertion of artificial miRNA into intronic region of an endogenous gene (for future gene therapy purpose). We successfully created all the mouse models required for these studies using i-PITT and Easi-CRISPR technologies. The detailed phenotypic analyses with these mice are currently in progress.

Academic Significance and Societal Importance of the Research Achievements

本研究は独自の遺伝子ノックダウン(機能の抑制)法、独自の遺伝子改変マウス作製法を応用した新たな実験系を提案するものであり、今回、その解析に有用なマウスモデルの開発に成功した。これにより、マウスの体内でより洗練された遺伝子発現のコントロールを行うことが可能となると期待される。それは、これまでに知られていなかった個体レベルでの遺伝子の機能を見出すことにも繋がるものであり、さらに最近話題となっているゲノム編集技術を応用し、将来的な新規遺伝子治療技術開発にも結びつくものと言える。

Report

(4 results)
  • 2018 Annual Research Report   Final Research Report ( PDF )
  • 2017 Annual Research Report
  • 2016 Annual Research Report
  • Research Products

    (24 results)

All 2018 2017 2016 Other

All Int'l Joint Research (3 results) Journal Article (6 results) (of which Int'l Joint Research: 4 results,  Peer Reviewed: 6 results,  Open Access: 5 results,  Acknowledgement Compliant: 1 results) Presentation (11 results) (of which Int'l Joint Research: 4 results,  Invited: 2 results) Book (1 results) Remarks (2 results) Patent(Industrial Property Rights) (1 results) (of which Overseas: 1 results)

  • [Int'l Joint Research] ジョージア大学(米国)

    • Related Report
      2018 Annual Research Report
  • [Int'l Joint Research] University of Nebraska, Medical Center(米国)

    • Related Report
      2017 Annual Research Report
  • [Int'l Joint Research] The University Of Nebraska(米国)

    • Related Report
      2016 Annual Research Report
  • [Journal Article] Intravenous Delivery of piggyBac Transposons as a Useful Tool for Liver-Specific Gene-Switching2018

    • Author(s)
      Nakamura Shingo、Ishihara Masayuki、Watanabe Satoshi、Ando Naoko、Ohtsuka Masato、Sato Masahiro
    • Journal Title

      International Journal of Molecular Sciences

      Volume: 19 Issue: 11 Pages: 3452-3452

    • DOI

      10.3390/ijms19113452

    • Related Report
      2018 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Isolation and Analysis of a Genome-Edited Single-Hepatocyte from a Cas9 Transgenic Mouse Line2018

    • Author(s)
      Sakurai Takayuki、Kamiyoshi Akiko、Ohtsuka Masato、Gurumurthy Channabasavaiah B.、Sato Masahiro、Shindo Takayuki
    • Journal Title

      Methods in Molecular Biology

      Volume: 1874 Pages: 257-271

    • DOI

      10.1007/978-1-4939-8831-0_15

    • ISBN
      9781493988303, 9781493988310
    • Related Report
      2018 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors2018

    • Author(s)
      Miura H, Quadros RM, Gurumurthy CB, Ohtsuka M
    • Journal Title

      Nat Protoc.

      Volume: 13 Issue: 1 Pages: 195-215

    • DOI

      10.1038/nprot.2017.153

    • Related Report
      2017 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Bone marrow-derived mesenchymal stem cells propagate immunosuppressive/anti-inflammatory macrophages in cell-to-cell contact-independent and -dependent manners under hypoxic culture2017

    • Author(s)
      Takizawa N, Okubo N, Kamo M, Chosa N, Mikami T, Suzuki K, Yokota S, Ibi M, Ohtsuka M, Taira M, Sasaki D, Yaegashi T, Ishisaki A, Kyakumoto S
    • Journal Title

      Experimental Cell Res.

      Volume: 358 Issue: 2 Pages: 411-420

    • DOI

      10.1016/j.yexcr.2017.07.014

    • Related Report
      2017 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins2017

    • Author(s)
      Quadros RM, Miura H, Harms DW, Akatsuka H, Sato T, Aida T, Redder R, Richardson GP, Inagaki Y, Sakai D, Buckley SM, Seshacharyulu P, Batra SK, Behlke MA, Zeiner SA, Jacobi AM, Izu Y, Thoreson WB, Urness LD, Mansour SL, Ohtsuka M, Gurumurthy CB
    • Journal Title

      Genome Biology

      Volume: 18 Issue: 1 Pages: 92-92

    • DOI

      10.1186/s13059-017-1220-4

    • Related Report
      2017 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Pronuclear Injection-based Targeted Transgenesis (PITT)2016

    • Author(s)
      Schilit S.L.P., Ohtsuka M., Quadros R. and Gurumurthy C.B.
    • Journal Title

      Curr. Protoc. Hum. Genet.

      Volume: 91 Issue: 1

    • DOI

      10.1002/cphg.23

    • Related Report
      2016 Annual Research Report
    • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
  • [Presentation] Development of a reporter mouse model suitable for evaluation of in vivo genome editing efficiency2018

    • Author(s)
      H Miura, CB Gurumurthy, M Ohtsuka
    • Organizer
      2018 Cold Spring Harbor meeting: Genome Engineering: The CRISPR/Cas Revolution
    • Related Report
      2018 Annual Research Report
    • Int'l Joint Research
  • [Presentation] ゲノム編集効率評価系モデルマウスの開発と評価2018

    • Author(s)
      三浦 浩美、佐藤 正宏、水谷 晃子、大塚 正人
    • Organizer
      第41回日本分子生物学会年会
    • Related Report
      2018 Annual Research Report
  • [Presentation] piggyBac系ベクターのハイドロダイナミクス遺伝子導入法はマウス肝臓での導入遺伝子の持続発現と肝細胞でのgene switchingを可能とする2018

    • Author(s)
      中村 伸吾、渡部 聡、石原 雅之、安藤 尚子、大塚 正人、佐藤 正宏
    • Organizer
      第41回日本分子生物学会年会
    • Related Report
      2018 Annual Research Report
  • [Presentation] CRISPR solution for creating Cre-LoxP genetics resources2017

    • Author(s)
      Rolen M Quadros, Hiromi Miura, Masato Ohtsuka, Donald W Harms, Channabasavaiah B Gurumurthy
    • Organizer
      Genome Engineering: The CRISPR-Cas Revolution(CSHL Meetings & Courses Program)
    • 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] 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] 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] Easi-CRISPR:高効率なノックインマウス作製法2016

    • Author(s)
      三浦 浩美、Gurumurthy Channabasavaiah、相田 知海、稲垣 豊、佐藤 正宏、佐藤 健人、大塚 正人
    • 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 Annual Research Report
  • [Presentation] Generation of knockdown mice using PITT and Easi-CRISPR2016

    • Author(s)
      Ohtsuka M.
    • Organizer
      MicroRNAs/Noncoding & Genome Engineering/Editing and Europe -2016
    • Place of Presentation
      Peterhouse University of Cambridge(Cambridge, UK)
    • Year and Date
      2016-11-01
    • Related Report
      2016 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] 長い一本鎖DNAを用いたノックインマウス作製2016

    • Author(s)
      三浦 浩美、Channabasavaiah Gurumurthy、稲垣 豊、大塚 正人
    • 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 Annual Research Report
  • [Presentation] Easi-CRISPR:長鎖一本鎖DNAを用いた高効率ノックイン法2016

    • Author(s)
      大塚正人,相田知海,三浦浩美,Channabasavaiah Gurumurthy
    • Organizer
      日本ゲノム編集学会第1回大会
    • Place of Presentation
      広島国際会議場(広島県広島市)
    • Year and Date
      2016-09-06
    • Related Report
      2016 Annual Research Report
  • [Book] PITT法によるターゲットトランスジェニックマウス作製 in 動物/疾患モデルの作製技術・病態解析・評価手法2017

    • Author(s)
      大塚正人、三浦浩美
    • Total Pages
      428
    • Publisher
      技術情報協会
    • ISBN
      9784861046650
    • Related Report
      2017 Annual Research 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 Annual Research Report
  • [Patent(Industrial Property Rights)] Dna editing using single-stranded dna2016

    • Inventor(s)
      C B GURU, H Miura, M Ohtsuka
    • Industrial Property Rights Holder
      The Univ. Nebraska, Tokai Univ.
    • Industrial Property Rights Type
      特許
    • Filing Date
      2016-06-03
    • Related Report
      2016 Annual Research Report
    • Overseas

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Published: 2016-04-21   Modified: 2022-05-20  

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