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Application of lipofection-mediated genome editing in mouse zygotes created by IVF via ultra-superovulation

Research Project

Project/Area Number 15H04606
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Integrative animal science
Research InstitutionKumamoto University

Principal Investigator

Nakagata Naomi  熊本大学, 生命資源研究・支援センター, 教授 (30159058)

Co-Investigator(Kenkyū-buntansha) 中川 佳子  熊本大学, 生命資源研究・支援センター, 特任助教 (30732739)
Co-Investigator(Renkei-kenkyūsha) Yamamoto Takashi  広島大学, 大学院理学研究科, 教授 (90244102)
Sakuma Tetsushi  広島大学, 大学院理学研究科, 講師 (90711143)
Project Period (FY) 2015-04-01 – 2018-03-31
Project Status Completed (Fiscal Year 2017)
Budget Amount *help
¥16,900,000 (Direct Cost: ¥13,000,000、Indirect Cost: ¥3,900,000)
Fiscal Year 2017: ¥4,290,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥990,000)
Fiscal Year 2016: ¥5,330,000 (Direct Cost: ¥4,100,000、Indirect Cost: ¥1,230,000)
Fiscal Year 2015: ¥7,280,000 (Direct Cost: ¥5,600,000、Indirect Cost: ¥1,680,000)
Keywordsマウス / ゲノム編集 / 受精卵 / 核酸導入 / 遺伝子 / 核酸 / ゲノム / 発生・分化 / バイオテクノロジー
Outline of Final Research Achievements

Recently, genome editing in mouse zygotes has become convenient and scalable, in association with various technological developments and improvements such as novel nuclease tools, alternative delivery methods, and contemporary reproductive engineering techniques. We have so far demonstrated the applicability of ultra-superovulation, in vitro fertilization (IVF), and vitrification/warming of zygotes in microinjection-mediated mouse genome editing. Here, we present an example of an application coupling the following three technologies: 1) CRISPR-Cas9 system as the most convenient genome-editing technology, 2) lipofection as the most simple, economical and effortless delivery method, and 3) cryopreserved oocytes created by IVF via ultra-superovulation as the most animal welfare- and user-friendly strategy. We successfully created gene knockout mice carrying insertion/deletion mutations.

Report

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

    (30 results)

All 2018 2017 2016 2015 Other

All Journal Article (8 results) (of which Int'l Joint Research: 2 results,  Peer Reviewed: 8 results,  Open Access: 7 results,  Acknowledgement Compliant: 2 results) Presentation (18 results) (of which Int'l Joint Research: 5 results,  Invited: 4 results) Book (1 results) Remarks (1 results) Patent(Industrial Property Rights) (2 results)

  • [Journal Article] History of cryobiology, with special emphasis in evolution of mouse sperm cryopreservation2018

    • Author(s)
      Sztein JM, Takeo T, Nakagata N
    • Journal Title

      Cryobiology

      Volume: 印刷中

    • Related Report
      2017 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Culture time of vitrified/warmed zygotes before microinjection affects the production efficiency of CRISPR-Cas9-mediated knock-in mice2017

    • Author(s)
      Nakagawa Y, Sakuma T, Nishimichi N, Yokosaki Y, Takeo T, Nakagata N, Yamamoto T
    • Journal Title

      Biology Open

      Volume: - Pages: 706-713

    • DOI

      10.1242/bio.025122

    • Related Report
      2017 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Fertility of cold-stored mouse sperm is recovered by promoting acrosome reaction and hyperactivation after cholesterol efflux by methyl-β-cyclodextrin2017

    • Author(s)
      Hidetaka Yoshimoto, Toru Takeo, Tetsumi Irie, Naomi Nakagata
    • Journal Title

      Biology of Reproduction

      Volume: 96 Issue: 2 Pages: 446-455

    • DOI

      10.1095/biolreprod.116.142901

    • Related Report
      2017 Annual Research Report
    • Peer Reviewed / Open Access / Acknowledgement Compliant
  • [Journal Article] Dimethyl sulfoxide and quercetin prolong the survival, motility, and fertility of cold-stored mouse sperm for 10 days2017

    • Author(s)
      Yoshimoto H, Takeo T, Nakagata N
    • Journal Title

      Biology of Reproduction

      Volume: 97 Issue: 6 Pages: 883-891

    • DOI

      10.1093/biolre/iox144

    • Related Report
      2017 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] バイオ研究施設の震災対策ー熊本地震における実験動物施設の被災・復旧からのレッスン2017

    • Author(s)
      中潟 直己
    • Journal Title

      実験医学

      Volume: 35 Pages: 1862-1866

    • Related Report
      2017 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Ultra-superovulation for the CRISPR-Cas9-mediated production of gene-knockout, single- amino-acid-substituted, and floxed mice2016

    • Author(s)
      Yoshiko Nakagawa, Tetsushi Sakuma, Norihisa Nishimichi, Yasuyuki Yokosaki, Noriyuki Yanaka, Toru Takeo, Naomi Nakagata and Takashi Yamamoto
    • Journal Title

      Biology Open

      Volume: 5 Issue: 8 Pages: 1142-1148

    • DOI

      10.1242/bio.019349

    • Related Report
      2016 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Superovulation Using the Combined Administration of Inhibin Antiserum and Equine Chorionic Gonadotropin Increases the Number of Ovulated Oocytes in C57BL/6 Female Mice2015

    • Author(s)
      Toru Takeo and Naomi Nakagata
    • Journal Title

      PLoS ONE

      Volume: 10 Issue: 5 Pages: 1-11

    • DOI

      10.1371/journal.pone.0128330

    • Related Report
      2015 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Production of knockout mice by DNA microinjection of various CRISPR/Cas9 vectors into freeze-thawed fertilized oocytes2015

    • Author(s)
      Nakagawa Y, Sakuma T, Sakamoto T, Ohmuraya M, Nakagata N, Yamamoto T
    • Journal Title

      BMC Biotechnol

      Volume: - Issue: 1 Pages: 33-33

    • DOI

      10.1186/s12896-015-0144-x

    • Related Report
      2015 Annual Research Report
    • Peer Reviewed / Open Access / Acknowledgement Compliant
  • [Presentation] AMED創薬基盤推進研究事業『マウスバンク機能の拡充による創薬イノベーションの迅速化』に関する取り組み2017

    • Author(s)
      竹尾 透、中川佳子、鳥越大輔、南 敬、杉本幸彦、中潟直己
    • Organizer
      日本薬学会第137年会
    • Place of Presentation
      仙台国際センター (宮城県仙台市)
    • Year and Date
      2017-03-24
    • Related Report
      2016 Annual Research Report
  • [Presentation] Culture time of zygotes before microinjection affects the production efficiency of CRISPR-Cas9-mediated knock-in mice2017

    • Author(s)
      Yoshiko Nakagawa, Tetsushi Sakuma, Norihisa Nishimichi, Yasuyuki Yokosaki, Toru Takeo, Naomi Nakagata, and Takashi Yamamoto
    • Organizer
      2nd annual Genome Editing & Engineering Conference
    • Related Report
      2017 Annual Research Report
    • Int'l Joint Research
  • [Presentation] CRISPR-Casシステムを用いたノックインマウス作製における凍結受精卵培養時間の検討2017

    • Author(s)
      中川佳子、佐久間哲史、西道教尚、横崎恭之、竹尾透、山本卓、中潟直己
    • Organizer
      第2回日本ゲノム編集学会
    • Related Report
      2017 Annual Research Report
  • [Presentation] ゲノム編集技術と生殖工学技術を用いた効率的な遺伝子改変マウス作製2017

    • Author(s)
      中川佳子、中潟直己
    • Organizer
      平成29年度 脳研究所共同利用共同研究 第1回合同セミナー
    • Related Report
      2017 Annual Research Report
  • [Presentation] CRISPR-Casシステムを用いたノックインマウス作製における凍結受精卵培養時間の検討2017

    • Author(s)
      中川佳子、佐久間哲史、西道教尚、横崎泰之、近藤朋子、竹下由美、中牟田裕子、 石田恵理、坂本亘、土山修治、竹尾 透、山本 卓、中潟直己
    • Organizer
      第35回動物生殖工学研究会
    • Related Report
      2017 Annual Research Report
  • [Presentation] CRISPR-Casシステムを用いたノックインマウス作製における凍結受精卵培養時間の検討2017

    • Author(s)
      中川佳子、佐久間哲史、西道教尚、横崎恭之、若松和子、春口幸恵、近藤朋子、竹下由美、中牟田裕子、石田恵理、坂本亘、土山修治、竹尾 透、山本 卓、中潟直己
    • Organizer
      第64回日本実験動物学会
    • Related Report
      2017 Annual Research Report
  • [Presentation] Establishment of an efficient mouse bank system using reproductive biotechnology, 3rd Oceania Mouse2017

    • Author(s)
      Toru Takeo and Naomi Nakagata
    • Organizer
      Transgenic/Assisted Reproductive Technology Symposium
    • Related Report
      2017 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] Introduction of CARD-TEXAS Mouse Reproductive Technology Workshop2017

    • Author(s)
      Naomi Nakagata
    • Organizer
      CARD-TEXAS Mouse Reproductive Technology Workshop
    • Related Report
      2017 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Creating a platform for mouse research using reproductive technology,2017

    • Author(s)
      Toru Takeo and Naomi Nakagata
    • Organizer
      2017 AMMRA & AMPC Meeting
    • Related Report
      2017 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] A Powerful Strategy To Conduct Mouse Research By Using The High-efficient Reproductive Technology2017

    • Author(s)
      Toru Takeo, Naomi Nakagata
    • Organizer
      Genome Editing USA Congress and co-located Advances in Transgenic Technology USA Congress
    • Related Report
      2017 Annual Research Report
    • Int'l Joint Research
  • [Presentation] 生殖工学技術の国内外の動向と3Rsの実践に向けたこれら技術の応用2017

    • Author(s)
      中潟直己
    • Organizer
      「特別講演」第51回日本実験動物技術者協会
    • Related Report
      2017 Annual Research Report
    • Invited
  • [Presentation] 生殖工学技術を駆使したマウスバンクシステム2017

    • Author(s)
      中潟 直己
    • Organizer
      「特別講演」日本婦人科腫瘍学会
    • Related Report
      2017 Annual Research Report
    • Invited
  • [Presentation] 遺伝子改変マウス・凍結胚/精子は、こうして守られた!2017

    • Author(s)
      中潟直己
    • Organizer
      第64回 日本実験動物学会
    • Related Report
      2017 Annual Research Report
  • [Presentation] 3Rs:啓蒙から実践へ―生殖工学技術の応用2017

    • Author(s)
      中潟直己
    • Organizer
      第64回日本実験動物学会
    • Related Report
      2017 Annual Research Report
  • [Presentation] CRISPR-Casシステムによるfloxマウスの作製-超過剰排卵誘起法を用いた凍結受精卵の利用-2016

    • Author(s)
      中川 佳子、佐久間 哲史、矢中 規之、山本 卓 、若松 和子 、山下 紀代子、春口 幸恵、近藤 朋子、竹下 由美、中牟田 裕子、梅野 智子、土山 修治 、竹尾  透 、中潟 直己
    • 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 Annual Research Report
  • [Presentation] CRISPR-Casシステムによる様々なゲノム編集個体の作製―超過剰排卵誘起法を用いた体外受精凍結卵 の利用2016

    • Author(s)
      中川佳子,佐久間哲史,西道教尚,横崎恭之,矢中規之,竹尾 透,中潟直己, 山本 卓
    • Organizer
      第一回ゲノム編集学会
    • 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
  • [Presentation] CRISPR-Casシステムによる遺伝子破壊マウスの作製-超過剰排卵誘起法を用いた凍結受精卵の利用-2016

    • Author(s)
      中川佳子、佐久間哲史、坂本拓弥、山本 卓、若松和子、山下紀代子、春口幸恵、近藤朋子、竹下由美、中牟田裕子、梅野智子、宮川 あい、土山修治、竹尾 透、中潟直己
    • Organizer
      第63回日本実験動物学会
    • 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 Annual Research Report
  • [Presentation] 凍結受精卵を用いたCRISPR/Cas9システムによる遺伝子破壊マウスの作製2015

    • Author(s)
      中川佳子、佐久間哲史、坂本拓弥、大村谷昌樹、山本卓、中潟直己
    • 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 Annual Research Report
  • [Book] マウス表現型解析スタンダード2016

    • Author(s)
      伊川正人、高橋智、若菜茂晴、中潟直己(第6章、第7章)
    • Total Pages
      347
    • Publisher
      羊土社
    • Related Report
      2016 Annual Research Report
  • [Remarks] 生命資源研究・支援センター 資源開発分野

    • URL

      http://irda.kuma-u.jp/divisions/reproductive_engineer/reproductive_engineer.html

    • Related Report
      2015 Annual Research Report
  • [Patent(Industrial Property Rights)] マウス精子の冷蔵保存液および保存方法2017

    • Inventor(s)
      竹尾 透、吉本英高、中潟直己
    • Industrial Property Rights Holder
      竹尾 透、吉本英高、中潟直己
    • Industrial Property Rights Type
      特許
    • Filing Date
      2017
    • Related Report
      2017 Annual Research Report
  • [Patent(Industrial Property Rights)] 新規過排卵誘起処理によるマウス卵子の大量作製法2015

    • Inventor(s)
      中潟直己、竹尾透
    • Industrial Property Rights Holder
      中潟直己、竹尾透
    • Industrial Property Rights Type
      特許
    • Filing Date
      2015-04-30
    • Acquisition Date
      2016-05-13
    • Related Report
      2016 Annual Research Report

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Published: 2015-04-16   Modified: 2019-03-29  

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