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Generation of gene modified mice using in situ genome editing system without microinjection

Research Project

Project/Area Number 19H03152
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Review Section Basic Section 42040:Laboratory animal science-related
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 医療工学研究部門, 教授 (00505323)

Co-Investigator(Kenkyū-buntansha) 佐藤 正宏  国立研究開発法人国立成育医療研究センター, ゲノム医療研究部, 共同研究員 (30287099)
大塚 正人  東海大学, 医学部, 教授 (90372945)
Project Period (FY) 2019-04-01 – 2022-03-31
Project Status Completed (Fiscal Year 2021)
Budget Amount *help
¥17,420,000 (Direct Cost: ¥13,400,000、Indirect Cost: ¥4,020,000)
Fiscal Year 2021: ¥5,460,000 (Direct Cost: ¥4,200,000、Indirect Cost: ¥1,260,000)
Fiscal Year 2020: ¥5,720,000 (Direct Cost: ¥4,400,000、Indirect Cost: ¥1,320,000)
Fiscal Year 2019: ¥6,240,000 (Direct Cost: ¥4,800,000、Indirect Cost: ¥1,440,000)
Keywords経胎盤的遺伝子導入(TPGD) / ゲノム編集 / 遺伝子改変マウス / 発生工学 / ソノポーレーション / in situ 遺伝子導入 / in situ遺伝子導入
Outline of Research at the Start

ゲノム編集技術の登場により、動物個体の遺伝子改変実験が盛んに行われている。主な実験方法はゲノム編集の因子を受精卵へ導入するものであり、多くの場合、受精卵の採取と遺伝子導入、受精卵の培養、受精卵の仮親への再移植といった高度で熟練した技術が必要である。本研究では、このような受精卵の操作を回避できる遺伝子改変動物作成のための簡便な新技術を検討する。本成果は、医学・獣医学・農学分野等で役立つ技術の確立につながるものと期待される。

Outline of Final Research Achievements

In this study, we demonstrated that intravenous injection of plasmid DNA containing CRISPR/Cas9 produced indels in fetal myocardial cells. This study has novelty, which is clearly different from other similar research including our previous study in view that: successful genome editing was achieved when hydrodynamics-based gene delivery was employed without using any gene delivery reagent or equipment, and mutations are induced successfully at the target endogenous MHCα locus. In addition, against our expectations, cells with genome editing were also found in non-cardiac cells. We have succeeded in creating convenient method of producing genome-modified fetuses using in situ genome editing system although there is still room for improvements to this method.

Academic Significance and Societal Importance of the Research Achievements

CRISPR/Cas9に代表されるゲノム編集技術により、動物個体の遺伝子改変技術が大きく進歩している。その中にあって本研究は、胚のex vivo操作に依らないin vivo遺伝子導入法によってCRISPR系を稼働させる点に特色を有する。本研究の成果は、心臓の内在性遺伝子が改変された胎仔が簡便に得られたことに集約される。今後、系の改良等本手法の検討をさらに進めていくことで遺伝子操作の対象となる組織が広がって行くものと考えられる。受精卵操作を一切回避したこの様な新技術の発展は、医学生物学分野の研究等に大きく貢献するものと期待できる。

Report

(4 results)
  • 2021 Annual Research Report   Final Research Report ( PDF )
  • 2020 Annual Research Report
  • 2019 Annual Research Report
  • Research Products

    (18 results)

All 2022 2021 2020 2019

All Journal Article (15 results) (of which Int'l Joint Research: 1 results,  Peer Reviewed: 11 results,  Open Access: 14 results) Presentation (2 results) Book (1 results)

  • [Journal Article] Electroporation-Based Non-Viral Gene Delivery to Adipose Tissue in Mice2022

    • Author(s)
      Masahiro Sato、Issei Saitoh、Yuki Kiyokawa、Eri Akasaka、Shingo Nakamura、Satoshi Watanabe、Emi Inada
    • Journal Title

      OBM Genetics

      Volume: 6 Issue: 2 Pages: 18-18

    • DOI

      10.21926/obm.genet.2202151

    • Related Report
      2021 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Recent Advances in the Production of Genome-Edited Rats2022

    • Author(s)
      Sato Masahiro、Nakamura Shingo、Inada Emi、Takabayashi Shuji
    • Journal Title

      International Journal of Molecular Sciences

      Volume: 23 Issue: 5 Pages: 2548-2548

    • DOI

      10.3390/ijms23052548

    • Related Report
      2021 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] ゲノム編集技術を利用した遺伝子改変マウスの簡便な作製法2021

    • Author(s)
      中村伸吾、秦 裕樹
    • Journal Title

      防衛医科大学校雑誌

      Volume: 46 Pages: 147-154

    • NAID

      40022803433

    • Related Report
      2021 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Hydrodynamics-based transplacental delivery as a useful noninvasive tool for manipulating fetal genome2020

    • Author(s)
      Nakamura S, Ando N, Watanabe S, Akasaka E, Ishihara M, Sato M
    • Journal Title

      Cells

      Volume: 9 Issue: 7 Pages: 1744-1744

    • DOI

      10.3390/cells9071744

    • Related Report
      2020 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Development of novel heparin/protamine nanoparticles useful for delivery of exogenous proteins in vitro and in vivo2020

    • Author(s)
      Nakamura S, Ando N, Ishihara M, Sato M
    • Journal Title

      Nanomaterials

      Volume: 10 Issue: 8 Pages: 1584-1584

    • DOI

      10.3390/nano10081584

    • Related Report
      2020 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] In vivo hepatocyte genome manipulation via intravenous injection of genome editing components2020

    • Author(s)
      Nakamura S, Ando N, Ishihara M, Sato M
    • Journal Title

      OBM Genetics

      Volume: 4 Issue: 4 Pages: 23-23

    • DOI

      10.21926/obm.genet.2004119

    • Related Report
      2020 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Conditional Liver-Specific Gene Expression System2020

    • Author(s)
      Nakamura Shingo
    • Journal Title

      Encyclopedia

      Volume: 2296 Pages: 1-4

    • Related Report
      2020 Annual Research Report
    • Open Access
  • [Journal Article] Transplacental Gene Delivery2020

    • Author(s)
      Nakamura Shingo
    • Journal Title

      Encyclopedia

      Volume: 407 Pages: 1-3

    • Related Report
      2020 Annual Research Report
    • Open Access
  • [Journal Article] Hydrodynamics-Based Transplacental Delivery of CRISPR/Cas92020

    • Author(s)
      Nakamura Shingo
    • Journal Title

      Encyclopedia

      Volume: 2197 Pages: 1-3

    • Related Report
      2020 Annual Research Report
    • Open Access
  • [Journal Article] Self-assembly-based manufacturing of nano-biomaterials2020

    • Author(s)
      Nakamura Shingo
    • Journal Title

      Encyclopedia

      Volume: 2297 Pages: 1-3

    • Related Report
      2020 Annual Research Report
    • Open Access
  • [Journal Article] piggyBac-Based Non-Viral In Vivo Gene Delivery Useful for Production of Genetically Modified Animals and Organs.2020

    • Author(s)
      Sato M, Inada E, Saitoh I, Watanabe S, Nakamura S.
    • Journal Title

      Pharmaceutics

      Volume: 12 Issue: 3 Pages: 277-277

    • DOI

      10.3390/pharmaceutics12030277

    • Related Report
      2019 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Recent Advances and Future Perspectives of In Vivo Targeted Delivery of Genome-Editing Reagents to Germ cells, Embryos, and Fetuses in Mice.2020

    • Author(s)
      Sato M, Takabayashi S, Akasaka E, Nakamura S.
    • Journal Title

      Cells

      Volume: 9 Issue: 4 Pages: 799-799

    • DOI

      10.3390/cells9040799

    • Related Report
      2019 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Potential for Isolation of Immortalized Hepatocyte Cell Lines by Liver-Directed In Vivo Gene Delivery of Transposons in Mice.2019

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

      Stem Cells International

      Volume: 2 Pages: 5129526-5129526

    • DOI

      10.1155/2019/5129526

    • Related Report
      2019 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] In Vivo Piggybac-Based Gene Delivery towards Murine Pancreatic Parenchyma Confers Sustained Expression of Gene of Interest.2019

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

      International Journal of Molecular Science

      Volume: 20 Issue: 13 Pages: 3116-3116

    • DOI

      10.3390/ijms20133116

    • Related Report
      2019 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Transplacental Gene Delivery (TPGD) as a Noninvasive Tool for Fetal Gene Manipulation in Mice2019

    • Author(s)
      Nakamura、Watanabe、Ando、Ishihara、Sato
    • Journal Title

      International Journal of Molecular Sciences

      Volume: 20 Issue: 23 Pages: 5926-5926

    • DOI

      10.3390/ijms20235926

    • Related Report
      2019 Annual Research Report
    • Peer Reviewed / Open Access
  • [Presentation] Transplacental delivery of CRISPR components via tail veins of pregnant females causes mutations in embryonic cardiomyocytes of murine fetuses.2019

    • Author(s)
      中村伸吾, 安藤尚子, 渡部聡, 石原雅之, 佐藤正宏.
    • Organizer
      第42回日本分子生物学会年会
    • Related Report
      2019 Annual Research Report
  • [Presentation] ブタ脂肪前駆細胞PSPAにおいて細胞分化に伴い発現が変化する遺伝子の網羅的解析.2019

    • Author(s)
      渡部聡, 櫻井敬之, 中村伸吾, 平岩秀樹, 土居考爾, 安江博, 佐藤正宏.
    • Organizer
      第42回日本分子生物学会年会
    • Related Report
      2019 Annual Research Report
  • [Book] Gene Editing - Technologies and Applications2019

    • Author(s)
      Sato M, Nakamura S.
    • Total Pages
      36
    • Publisher
      IntechOpen
    • Related Report
      2019 Annual Research Report

URL: 

Published: 2019-04-18   Modified: 2023-01-30  

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