Development versatile zygote-mediated genome-editing technologies in mice

• Project/Area Number: 26712025
• Research Category: Grant-in-Aid for Young Scientists (A)
• Allocation Type: Partial Multi-year Fund
• Research Field: Integrative animal science
• Research Institution: The University of Tokyo
• Principal Investigator: Fujii Wataru 東京大学, 大学院農学生命科学研究科(農学部), 助教 (40708161)
• Project Period (FY): 2014-04-01 – 2018-03-31
• Project Status: Completed (Fiscal Year 2017)
• Budget Amount: ¥23,790,000 (Direct Cost: ¥18,300,000、Indirect Cost: ¥5,490,000); Fiscal Year 2017: ¥4,030,000 (Direct Cost: ¥3,100,000、Indirect Cost: ¥930,000); Fiscal Year 2016: ¥4,030,000 (Direct Cost: ¥3,100,000、Indirect Cost: ¥930,000); Fiscal Year 2015: ¥6,240,000 (Direct Cost: ¥4,800,000、Indirect Cost: ¥1,440,000); Fiscal Year 2014: ¥9,490,000 (Direct Cost: ¥7,300,000、Indirect Cost: ¥2,190,000)
• Keywords: 発生工学 / 人工ヌクレアーゼ / ゲノム改変動物 / 構築遺伝学

Outline of Final Research Achievements

We attempted to establish more versatile system to generate genetically-modified mice by using zygote-mediated engineered endonucleases. We were successfully applied S. thermophiles 1- and C. jejuni-derived CRISPR/Cas to zygote-mediated generation of knockout/knock-in mice. We found that exogenous dsDNA-injection activates ooplasmic ATM signaling pathway and inhibits preimplantation development. By using the established method, we generated several mouse strains which replaced the orthologous locus/loci to porcine or giraffe gene sequence.

Research Products

• [Journal Article] MRP14 is dispensable for LPS-induced shock in BALB/c mice (2018)
  • Author(s): H. Mizobuchi, W. Fujii, K. Ishizuka, Y. Wang, S. Watanabe, C. Sanjoba, Y. Matsumoto, Y. Goto
  • Journal Title: Immunology Letters Volume: 194 Pages: 13-20
  • DOI: 10.1016/j.imlet.2017.12.003
  • Peer Reviewed
• [Journal Article] Generation of Knock-in Mouse by Genome Editing. (2017)
  • Author(s): Fujii W
  • Journal Title: Methods in Molecular Biology Volume: 1630 Pages: 91-100
  • DOI: 10.1007/978-1-4939-7128-2_8
  • ISBN: 9781493971275, 9781493971282
  • Peer Reviewed
• [Journal Article] Efficient generation of genome-modified mice using Campylobacter jejuni-derived CRISPR/Cas. (2017)
  • Author(s): Fujii W, Ikeda A, Sugiura K, Naito K.
  • Journal Title: International Jornal of Molecular Sciences Volume: 18 Issue: 11 Pages: 2286-2286
  • DOI: 10.3390/ijms18112286
  • Peer Reviewed / Open Access
• [Journal Article] The essential role of phospho-T38 CPI-17 in the maintenance of physiological blood pressure using genetically modified mice (2017)
  • Author(s): Yang Q, Fujii W, Kaji N, Kakuta S, Kada K, Kuwahara M, Tsubone H, Ozaki H, Hori M
  • Journal Title: FASEB J Volume: 32(4) Issue: 4 Pages: 2095-2109
  • DOI: 10.1096/fj.201700794r
  • Peer Reviewed / Open Access
• [Journal Article] B-cell activating factor deficiency suppresses splenomegaly during Leishmania donovani infection. (2017)
  • Author(s): Omachi S, Fujii W, Azuma N, Morimoto A, Sanjoba C, Matsumoto Y, Goto Y.
  • Journal Title: Biochem Biophys Res Commun. Volume: 489(4) Issue: 4 Pages: 528-533
  • DOI: 10.1016/j.bbrc.2017.06.005
  • Peer Reviewed
• [Journal Article] Efficient mutagenesis by CRISPR/Cas system during meiotic maturation of porcine oocytes (2017)
  • Author(s): Onuma A, Fujii W, Sugiura K, Naito K.
  • Journal Title: Journal of Reproduction and Development Volume: 63 Issue: 1 Pages: 45-50
  • DOI: 10.1262/jrd.2016-094
  • NAID: 130005332294
  • ISSN: 0916-8818, 1348-4400
  • Peer Reviewed / Open Access
• [Journal Article] Recent biotechnology tools contributing to the molecular-genetics analysis for non-model animals. (2017)
  • Author(s): 藤井渉
  • Journal Title: The Journal of Animal Genetics Volume: 45 Issue: 1 Pages: 19-30
  • DOI: 10.5924/abgri.45.19
  • NAID: 130005440027
  • ISSN: 1345-9961, 1884-3883
  • Peer Reviewed / Open Access
• [Journal Article] Zygote-mediated generation of genome-modified mice using Streptococcus thermophiles 1-derived CRISPR/Cas system. (2016)
  • Author(s): Fujii W, Kakuta S, Yoshioka S, Kyuwa S,Sugiura K,Naito K.
  • Journal Title: Biochemical and Biophysical Research Communications Volume: 477 Issue: 3 Pages: 473-476
  • DOI: 10.1016/j.bbrc.2016.06.070
  • Peer Reviewed / Open Access
• [Journal Article] 農学的利用に向けたゲノム編集の現状・将来の展望 (2016)
  • Author(s): 藤井渉
  • Journal Title: 化学と生物 Volume: 54 Pages: 568-574
  • NAID: 130005815185
  • Peer Reviewed
• [Journal Article] CRISPR/Casシステムの近年の動向とインターフェロン・サイトカイン分野への応用 (2016)
  • Author(s): 藤井渉, 角田茂
  • Journal Title: JSICR Newsletter Volume: 41 Pages: 8-14
  • Peer Reviewed / Open Access
• [Journal Article] Finding of a highly efficient ZFN pair for <i>Aqpep</i> gene functioning in murine zygotes (2015)
  • Author(s): Fujii W, Onuma A, Yoshioka S, Nagashima K, Sugiura K, Naito K
  • Journal Title: Journal of Reproduction and Development Volume: 61 Issue: 6 Pages: 589-593
  • DOI: 10.1262/jrd.2015-087
  • NAID: 130005113567
  • ISSN: 0916-8818, 1348-4400
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Development of a mono-promoter-driven CRISPR/Cas9 system in mammalian cells (2015)
  • Author(s): Shin Yoshioka, Wataru Fujii, Tetsuhiro Ogawa, Koji Sugiura, and Kunihiko Naito
  • Journal Title: Scientific Reports Volume: 5 Issue: 1 Pages: 18341-18341
  • DOI: 10.1038/srep18341
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Tetraploid embryonic stem cells maintain pluripotency and differentiation potency into three germ layers (2015)
  • Author(s): Imai H, Kano K*, Fujii W, Takasawa K, Wakitani S, Hiyama M, Nishino K, Kusakabe KT, Kiso Y
  • Journal Title: PLoS ONE Volume: 10 Issue: 6 Pages: e0130585-e0130585
  • DOI: 10.1371/journal.pone.0130585
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] マウス個体でのゲノム編集(1)―効率よく正確なゲノム改変を施すには (2015)
  • Author(s): 藤井渉
  • Journal Title: 医学のあゆみ Volume: 252 Pages: 159-163
• [Journal Article] マウス個体でのゲノム編集（１）――マウス個体でのゲノム編集(1)―効率よく正確なゲノム改変を施すには. (2015)
  • Author(s): 藤井渉.
  • Journal Title: 医学のあゆみ Volume: 252(2) Pages: 159-163
• [Journal Article] One-step Generation of Phenotype-expressing Triple-knockout Mice with Heritable Mutated Alleles by the CRISPR/Cas9 System (2014)
  • Author(s): Fujii W, Onuma A, Sugiura K, Naito K.
  • Journal Title: Journal of Reproduction and Development Volume: 60 Issue: 4 Pages: 324-327
  • DOI: 10.1262/jrd.2013-139
  • NAID: 130004137824
  • ISSN: 0916-8818, 1348-4400
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Nakamura K, Fujii W, Tsuboi M, Tanihata J, Teramoto N, Takeuchi S, Naito K, Yamanouchi K, Nishihara M (2014)
  • Author(s): Nakamura K, Fujii W, Tsuboi M, Tanihata J, Teramoto N, Takeuchi S, Naito K, Yamanouchi K, Nishihara M
  • Journal Title: Scientific Reports Volume: 4 Issue: 1 Pages: 5635-5635
  • DOI: 10.1038/srep05635
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] CRISPR/Cas RNA注入によるゲノム改変マウスの作製 (2014)
  • Author(s): 藤井渉
  • Journal Title: JSI Newsletter Volume: 22(2) Pages: 29-29
  • Open Access
• [Journal Article] ノックアウトマウスの基礎と応用. (2014)
  • Author(s): 角田茂、藤井渉.
  • Journal Title: 小児外科 Volume: 46(6): Pages: 567-570
• [Journal Article] 遺伝子改変動物の最新事情. (2014)
  • Author(s): 角田茂、藤井渉.
  • Journal Title: LABIO21 Volume: 57 Pages: 16-19
  • NAID: 40020154506
• [Journal Article] ゲノム編集へのいざない-培養細胞・マウス個体でのゲノム編集入門. (2014)
  • Author(s): 藤井渉.
  • Journal Title: JSICR Newsletter Volume: 38 Pages: 36-42
• [Presentation] Generation of genome-modified animals using engineered endonucleases (2017)
  • Author(s): Fujii W
  • Organizer: International Seminar "Insulin-like Signaling and Nutrient Signaling: universal signaling for extension of helthy lifeplan and improvemnt of quality for human and animals"
  • Place of Presentation: 東京大学フードサイエンス棟 （東京都文京区）
  • Year and Date: 2017-01-24
  • Int'l Joint Research / Invited
• [Presentation] ゲノム編集技術の現状と疾患モデル動物作製への応用 (2017)
  • Author(s): 藤井渉
  • Organizer: Basic Biology Seminar In Okayama
  • Invited
• [Presentation] ゲノム編集技術の最前線と応用 (2017)
  • Author(s): 藤井渉
  • Organizer: 第9回生殖補助医療胚培養士セミナー
  • Invited
• [Presentation] 次世代の応用遺伝学に向けた取り組み (2016)
  • Author(s): 藤井渉
  • Organizer: 東京大学優駿会
  • Place of Presentation: 東京大学フードサイエンス棟 （東京都文京区）
  • Invited
• [Presentation] 人工ヌクレアーゼを応用したゲノム改変動物作製技術の開発 (2016)
  • Author(s): 藤井渉
  • Organizer: 第15回日本農学進歩賞受賞講演
  • Place of Presentation: 東京大学弥生講堂 （東京都文京区）
  • Invited
• [Presentation] 退学後に考えたこと (2016)
  • Author(s): 藤井渉
  • Organizer: 岡山大学農学部同窓会セミナー
  • Place of Presentation: 岡山大学農学部同窓会
  • Invited
• [Presentation] ゲノム編集による遺伝子組換え動物作製の基礎と応用 (2016)
  • Author(s): 藤井渉
  • 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）
  • Invited
• [Presentation] 動物ゲノム編集研究の現状と展望 (2015)
  • Author(s): 藤井渉
  • Organizer: 第91回実験動物コンファレンス
  • Place of Presentation: 日本獣医生命科学大学（東京都・武蔵野市）
  • Year and Date: 2015-12-12
  • Invited
• [Presentation] ゲノム編集技術による遺伝子組換え動物作出の現状と展望 (2015)
  • Author(s): 藤井渉
  • Organizer: 京都大学霊長類研究所&中部幹細胞クラブ 研究会 第二回「霊長類への展開に向けた幹細胞・発生・エピゲノム研究」
  • Place of Presentation: 京都大学霊長類研究所（愛知県・犬山市）
  • Year and Date: 2015-09-01
  • Invited
• [Presentation] CRISPR/CASシステムを利用した遺伝子改変マウス作製の実際 (2015)
  • Author(s): 藤井渉
  • Organizer: 東京大学 医科学研究所
  • Place of Presentation: 東京大学 医科学研究所（東京都・港区）
  • Year and Date: 2015-07-31
  • Invited
• [Presentation] 人工ヌクレアーゼによるゲノム改変動物作製法の現状と展望 (2015)
  • Author(s): 藤井渉
  • Organizer: 名古屋大学 農学部
  • Place of Presentation: 名古屋大学 農学部（愛知県・名古屋市）
  • Year and Date: 2015-06-26
  • Invited
• [Presentation] ゲノム編集による遺伝子組換え動物作製の現状と課題 (2015)
  • Author(s): 藤井渉
  • 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: 2015-05-29
  • Invited
• [Presentation] ドナーDNA の状態がCRISPR/Casを用いたマウス前核期卵へのノックインに及ぼす影響 (2015)
  • Author(s): 永島圭介、藤井渉、杉浦幸二、内藤邦彦
  • Organizer: 日本畜産学会
  • Place of Presentation: 宇都宮大学
  • Year and Date: 2015-03-28
• [Presentation] 人工ヌクレアーゼによるゲノム改変動物の作製 (2014)
  • Author(s): 藤井渉
  • Organizer: 細胞センサーの分子機構・相互関連・ネットワーク研究会
  • Place of Presentation: 生理学研究所
  • Year and Date: 2014-12-04
  • Invited
• [Presentation] CRISPR/Casシステムを利用したゲノム編集 (2014)
  • Author(s): 藤井渉
  • Organizer: 「細胞を創る」研究会 7.0
  • Place of Presentation: 東京大学
  • Year and Date: 2014-11-14
  • Invited
• [Presentation] 人工ヌクレアーゼによるゲノム改変動物の作製 (2014)
  • Author(s): 藤井渉
  • Organizer: 特別セミナー
  • Place of Presentation: 東京大学農学部
  • Year and Date: 2014-07-18
  • Invited
• [Presentation] 人工ヌクレアーゼによるゲノム改変動物の作製 (2014)
  • Author(s): 藤井渉
  • Organizer: 特別セミナー
  • Place of Presentation: 農業生物資源研究所
  • Year and Date: 2014-07-15
  • Invited
• [Presentation] 人工ヌクレアーゼによるゲノム改変動物の作製 (2014)
  • Author(s): 藤井渉
  • Organizer: 特別セミナー
  • Place of Presentation: 東北大学農学部
  • Year and Date: 2014-06-03
  • Invited
• [Presentation] Generation of genome-modified animals usign CRISPR/Cas system (2014)
  • Author(s): 藤井渉
  • Organizer: 特別セミナー
  • Place of Presentation: Tzu Chi University, Taiwan
  • Year and Date: 2014-05-28
  • Invited
• [Presentation] Generation of genome-modified animals usign CRISPR/Cas system (2014)
  • Author(s): 藤井渉
  • Organizer: 特別セミナー
  • Place of Presentation: Institute of Molecular Biology, Academia Sinica, Taiwan
  • Year and Date: 2014-05-27
  • Invited
• [Presentation] How to Generate the genome-modified animals usign CRISPR/Cas system (2014)
  • Author(s): 藤井渉
  • Organizer: 特別セミナー
  • Place of Presentation: Institute of Molecular Biology, Academia Sinica, Taiwan
  • Year and Date: 2014-05-27
  • Invited
• [Book] 進化するゲノム編集技術 (2015)
  • Author(s): 藤井渉
  • Total Pages: 386
  • Publisher: エヌ・ティー・エス
• [Remarks] 藤井渉個人ホームページ
  • URL: http://wtrfujii.com/
• [Remarks] 東京大学大学院農学生命科学研究科応用遺伝学研究室ホームページ
  • URL: http://www.vm.a.u-tokyo.ac.jp/iden/
• [Remarks] 東京大学農学部 応用遺伝学研究室ホームページ
  • URL: http://www.vm.a.u-tokyo.ac.jp/iden/mokuji.htm
• [Remarks] 東京大学大学院農学生命科学研究科応用動物科学専攻応用遺伝学研究室ホームページ
  • URL: http://www.vm.a.u-tokyo.ac.jp/iden/
• [Remarks] 藤井渉ホームページ
  • URL: http://wtrfujii.com/
• [Remarks] 東京大学大学院農学生命科学研究科応用遺伝学研究室ホームページ
  • URL: http://www.vm.a.u-tokyo.ac.jp/iden/ouyouiden/publication.htm