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Development of microinjection-independent animal genome manipulation method

Research Project

Project/Area Number 16H05049
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Integrative animal science
Research InstitutionNational Defense Medical College

Principal Investigator

NAKAMURA Shingo  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, 講師 (00505323)

Co-Investigator(Kenkyū-buntansha) 石原 雅之  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, 教授 (10508500)
佐藤 正宏  鹿児島大学, 総合科学域総合研究学系, 教授 (30287099)
大塚 正人  東海大学, 医学部, 准教授 (90372945)
Project Period (FY) 2016-04-01 – 2019-03-31
Project Status Completed (Fiscal Year 2018)
Budget Amount *help
¥17,160,000 (Direct Cost: ¥13,200,000、Indirect Cost: ¥3,960,000)
Fiscal Year 2018: ¥5,330,000 (Direct Cost: ¥4,100,000、Indirect Cost: ¥1,230,000)
Fiscal Year 2017: ¥5,590,000 (Direct Cost: ¥4,300,000、Indirect Cost: ¥1,290,000)
Fiscal Year 2016: ¥6,240,000 (Direct Cost: ¥4,800,000、Indirect Cost: ¥1,440,000)
Keywords発生工学 / ゲノム編集 / 遺伝子改変マウス / 遺伝子導入
Outline of Final Research Achievements

The traditional transgenic techniques based on microinjection process, require expensive micromanipulator systems and a high level of skill to operate the equipment. In recently, sequence specific nucleases have discovered and revolutionized in the field of developmental engineering. In particular, the CRISPR/Cas9 system has emerged as a very simple and efficient approach to create genome edited mice. Based on such backgrounds, in this study, we attempt to develop microinjection-independent animal genome manipulation method that do not need isolation and ex vivo handling steps but that can deliver the CRISPR/Cas9 system directly in situ. As results, we achieved to elicit CRISPR/Cas9-mediated mutations in a target locus of embryonic cells by intravenous introducing a single plasmid conferring expression of both Cas9 and gRNA via pregnant mice. This may be a useful tool for manipulating the function of embryonic cells in vivo, allowing to create transgenic animal simply.

Academic Significance and Societal Importance of the Research Achievements

遺伝子改変動物の作製では、多くの場合、高価な機材とそれを操作する高度な技術等が必要になる。本研究の目的は、それらを回避できる簡便な新技術を検討することである。その成果は、遺伝子関連の研究を行っている多くの研究者にとって有用なものとなり、特に獣医学、農学、医学分野において貢献できると期待される。また、使用する動物個体数の削減による動物福祉の向上についても期待が持てる。

Report

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

    (27 results)

All 2019 2018 2017 2016

All Journal Article (16 results) (of which Int'l Joint Research: 2 results,  Peer Reviewed: 16 results,  Open Access: 14 results,  Acknowledgement Compliant: 1 results) Presentation (10 results) Book (1 results)

  • [Journal Article] Transplacental Delivery of Genome Editing Components Causes Mutations in Embryonic Cardiomyocytes of Mid-Gestational Murine Fetuses2019

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

      IUBMB Life

      Volume: 印刷中 Issue: 7 Pages: 835-844

    • DOI

      10.1002/iub.2004

    • Related Report
      2018 Annual Research Report
    • Peer Reviewed / Open Access
  • [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] The Combinational Use of CRISPR/Cas9 and Targeted Toxin Technology Enables Efficient Isolation of Bi-Allelic Knockout Non-Human Mammalian Clones2018

    • Author(s)
      Watanabe Satoshi、Sakurai Takayuki、Nakamura Shingo、Miyoshi Kazuchika、Sato Masahiro
    • Journal Title

      International Journal of Molecular Sciences

      Volume: 19 Issue: 4 Pages: 1075-1075

    • DOI

      10.3390/ijms19041075

    • Related Report
      2018 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] In vivo genome editing targeted towards the female reproductive system2018

    • Author(s)
      Sato Masahiro、Ohtsuka Masato、Nakamura Shingo、Sakurai Takayuki、Watanabe Satoshi、Gurumurthy Channabasavaiah B.
    • Journal Title

      Archives of Pharmacal Research

      Volume: 41 Issue: 9 Pages: 898-910

    • DOI

      10.1007/s12272-018-1053-z

    • Related Report
      2018 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [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] Timing of CRISPR/Cas9-related mRNA microinjection after activation as an important factor affecting genome editing efficiency in porcine oocytes2018

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

      Theriogenology

      Volume: 108 Pages: 29-38

    • DOI

      10.1016/j.theriogenology.2017.11.030

    • NAID

      120007099790

    • Related Report
      2017 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Protective effect of FGF-2 and low-molecular-weight heparin/protamine nanoparticles on radiation-induced healing-impaired wound repair in rats2017

    • Author(s)
      Kinoda Jun、Ishihara Masayuki、Nakamura Shingo、Fujita Masanori、Fukuda Koichi、Sato Yoko、Yokoe Hidetaka
    • Journal Title

      Journal of Radiation Research

      Volume: 59 Issue: 1 Pages: 27-34

    • DOI

      10.1093/jrr/rrx044

    • Related Report
      2017 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Efficient Generation of Somatic Cell Nuclear Transfer-Competent Porcine Cells with Mutated Alleles at Multiple Target Loci by Using CRISPR/Cas9 Combined with Targeted Toxin-Based Selection System.2017

    • Author(s)
      Sato M, Miyoshi K, Nakamura S, Ohtsuka M, Sakurai T, Watanabe S, Kawaguchi H, Tanimoto A.
    • Journal Title

      Int J Mol Sci

      Volume: 18 Issue: 12 Pages: 2610-2610

    • DOI

      10.3390/ijms18122610

    • NAID

      120007099791

    • Related Report
      2017 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Stability of Weakly Acidic Hypochlorous Acid Solution with Microbicidal Activity2017

    • Author(s)
      Ishihara Masayuki、Murakami Kaoru、Fukuda Koichi、Nakamura Shingo、Kuwabara Masahiro、Hattori Hidemi、Fujita Masanori、Kiyosawa Tomoharu、Yokoe Hidetaka
    • Journal Title

      Biocontrol Science

      Volume: 22 Issue: 4 Pages: 223-227

    • DOI

      10.4265/bio.22.223

    • NAID

      130006285032

    • ISSN
      1342-4815, 1884-0205
    • Related Report
      2017 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Cleansing technique using high-velocity steam?air micromist jet spray2017

    • Author(s)
      Fukuda Koichi、Ishihara Masayuki、Murakami Kaoru、Nakamura Shingo、Sato Yoko、Kuwabara Masahiro、Fujita Masanori、Kiyosawa Tomoharu、Yokoe Hidetaka
    • Journal Title

      Journal of Medical Engineering & Technology

      Volume: 41 Issue: 7 Pages: 522-528

    • DOI

      10.1080/03091902.2017.1364309

    • Related Report
      2017 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Intrapancreatic Parenchymal Injection of Cells as a Useful Tool for Allowing a Small Number of Proliferative Cells to Grow In Vivo.2017

    • Author(s)
      Sato M, Saitoh I, Murakami T, Kubota N, Nakamura S, Watanabe S, Inada E
    • Journal Title

      International Journal of Molecular Sciences

      Volume: 18 Issue: 8 Pages: 1678-1678

    • DOI

      10.3390/ijms18081678

    • Related Report
      2017 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Improved Survival of Full-Thickness Skin Graft With Low-Molecular Weight Heparin-Protamine Micro/Nanoparticles Including Platelet-Rich Plasma2017

    • Author(s)
      Takabayashi Yuki、Ishihara Masayuki、Kuwabara Masahiro、Takikawa Makoto、Nakamura Shingo、Hattori Hidemi、Kiyosawa Tomoharu
    • Journal Title

      Annals of Plastic Surgery

      Volume: 78 Issue: 5 Pages: 562-568

    • DOI

      10.1097/sap.0000000000001051

    • Related Report
      2017 Annual Research Report
    • Peer Reviewed
  • [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 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Low-molecular weight heparin protamine complex augmented the potential of adipose-derived stromal cells to ameliorate limb ischemia2016

    • Author(s)
      Satoko Kishimoto, Ken-ichi Inoue, Shingo Nakamura, Hidemi Hattori, Masayuki Ishihara, Masashi Sakuma, Shigeru Toyoda, Hideki Iwaguro, Isao Taguchi, Teruo Inoue, Ken-ichiro Yoshida
    • Journal Title

      Atherosclerosis

      Volume: 249 Pages: 132-139

    • DOI

      10.1016/j.atherosclerosis.2016.04.003

    • Related Report
      2016 Annual Research Report
    • Peer Reviewed / Open Access / Acknowledgement Compliant
  • [Journal Article] Cytotoxicity of Silver Nanoparticle and Chitin-Nanofiber Sheet Composites Caused by Oxidative Stress.2016

    • Author(s)
      Kinoda J, Ishihara M, Hattori H, Nakamura S, Fukuda K, Yokoe H.
    • Journal Title

      Nanomaterials (Basel).

      Volume: 6 Issue: 10 Pages: 189-189

    • DOI

      10.3390/nano6100189

    • Related Report
      2016 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Enhanced effect of fibroblast growth factor-2-containing dalteparin/protamine nanoparticles on hair growth.2016

    • Author(s)
      Takabayashi Y, Nambu M, Ishihara M, Kuwabara M, Fukuda K, Nakamura S, Hattori H, Kiyosawa T.
    • Journal Title

      Clin Cosmet Investig Dermatol.

      Volume: 9 Pages: 127-134

    • DOI

      10.2147/ccid.s108187

    • Related Report
      2016 Annual Research Report
    • Peer Reviewed
  • [Presentation] 体細胞核移植胚への直接 electroporation(GENTEP)は効率的なゲノム編集ブタ作製に有効である2018

    • Author(s)
      佐藤正宏, 三好和睦, 松永将伍, 渡部聡, 中村伸吾, 川口博明, 谷本昭英
    • Organizer
      第41回日本分子生物学会年会
    • Related Report
      2018 Annual Research Report
  • [Presentation] 卵管内注入によるゲノム編集動物作製法(GONAD)の改良:性腺刺激ホルモン投与による実験時間の制御2018

    • Author(s)
      佐藤正宏, 大塚正人, 中村伸吾
    • Organizer
      第111回日本繁殖生物学会大会
    • Related Report
      2018 Annual Research Report
  • [Presentation] 少数個の増殖性細胞の体内増殖を可能とする新規マウス膵臓内細胞移植法2017

    • Author(s)
      佐藤 正宏,齋藤一誠,村上智哉,窪田直子,中村伸吾,渡部聡,稲田絵美.
    • Organizer
      第40回日本分子生物学会年会
    • Related Report
      2017 Annual Research Report
  • [Presentation] 卵管内受精卵を標的とした生体内ゲノム編集法(GONAD)習得のための2日間プロトコール2017

    • Author(s)
      大塚正人,中村伸吾,CB. Gurumurthy,Naomi Arifin,Md Atiqul Islam,三浦浩美,佐藤正宏.
    • Organizer
      第51回日本実験動物技術者協会総会
    • Related Report
      2017 Annual Research Report
  • [Presentation] 銀ナノ粒子含有複合材料による殺微生物・ウイルス不活化効果2017

    • Author(s)
      中村伸吾,佐藤洋子,桑原征宏,福田孝一,安藤尚子,後藤寿美代,藤田真敬,石原雅之.
    • Organizer
      日本防菌防黴学会 第44回年次大会
    • Related Report
      2017 Annual Research Report
  • [Presentation] 水蒸気・空気混相マイクロミストジェット噴射による洗浄―その医療・獣医療応用への基礎的研究2017

    • Author(s)
      福田孝一,中村伸吾,佐藤洋子,後藤寿美代,安藤尚子,藤田真敬,石原雅之.
    • Organizer
      日本防菌防黴学会 第44回年次大会
    • Related Report
      2017 Annual Research Report
  • [Presentation] 大腸菌群および一般生菌の増殖と光照射による飲料水中の亜硝酸態窒素の挙動分析2017

    • Author(s)
      佐藤洋子,中村伸吾,後藤寿美代,安藤尚子,福田孝一,藤田真敬,石原雅之.
    • Organizer
      日本防菌防黴学会 第44回年次大会
    • Related Report
      2017 Annual Research Report
  • [Presentation] マウスすい臓内への piggyBac系を介した直接生体内遺伝子導入は外来遺伝子の長期発現を可能とする2016

    • Author(s)
      佐藤正宏, 稲田絵美, 齋藤一誠, 三浦浩美, 大塚正人, 中村伸吾, 桜井敬之, 渡部聡.
    • Organizer
      第39回日本分子生物学会年会
    • Place of Presentation
      パシフィコ横浜
    • Year and Date
      2016-11-30
    • Related Report
      2016 Annual Research Report
  • [Presentation] piggyBacベクターシステムとin vivo gene transferを用いた新たな肝細胞株樹立の試み2016

    • Author(s)
      渡部聡, 中村伸吾, 桜井敬之, 大塚正人, 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
    • Year and Date
      2016-11-30
    • Related Report
      2016 Annual Research Report
  • [Presentation] piggyBac遺伝子導入系により複数遺伝子が同時導入されたブタ細胞は核移植後のクローン胚の発生を保証する2016

    • Author(s)
      佐藤正宏, 前田昂亮, 郡山実優, 稲田絵美, 齋藤一誠, 大塚正人, 中村伸吾, 桜井敬之, 渡部聡, 三好和睦.
    • Organizer
      第109回日本繁殖生物学会大会
    • Place of Presentation
      麻布大学
    • Year and Date
      2016-09-11
    • Related Report
      2016 Annual Research Report
  • [Book] New Insights into Theriogenology2018

    • Author(s)
      Sato Masahiro、Ohtsuka Masato、Nakamura Shingo
    • Publisher
      IntechOpen
    • ISBN
      9781789847734
    • Related Report
      2018 Annual Research Report

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Published: 2016-04-21   Modified: 2020-03-30  

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