Production of immunodeficient micro-mini pig using genome editing technology.

• Project/Area Number: 18K12062
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 90110:Biomedical engineering-related
• Research Institution: The University of Tokushima
• Principal Investigator: HIRATA Maki 徳島大学, 大学院社会産業理工学研究部(生物資源産業学域), 講師 (10815583)
• Co-Investigator(Kenkyū-buntansha): 音井 威重 徳島大学, 大学院社会産業理工学研究部(生物資源産業学域), 教授 (30311814) ; 谷原 史倫 自治医科大学, 先端医療技術開発センター, 准教授 (90754680)
• Project Period (FY): 2018-04-01 – 2021-03-31
• Project Status: Completed (Fiscal Year 2020)
• Budget Amount: ¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000); Fiscal Year 2020: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2019: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2018: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
• Keywords: ゲノム編集 / ブタ / 免疫不全 / ミニブタ / マイクロミニブタ

Outline of Final Research Achievements

Pigs are useful research model animals that show many characteristics similar to humans. We aimed to produce immunodeficient micro-mini pigs using genome editing technology. In this study, we achieved in vitro fertilization using semen collected from a commercial micro mini pig. In addition, we demonstrated the feasibility of the introduction of double biallelic mutation of the two target genes (IL2RG and GHR), using electroporation to transfer two gRNAs and Cas9 protein to in vitro fertilized embryos. Hence, it will be possible to produce mico-mini-sized immunodeficient pigs by the combination of these technologies.

Academic Significance and Societal Importance of the Research Achievements

近年、iPS細胞をはじめとする幹細胞を用いた再生医療研究は、従来の方法では治療できない難治性疾患への臨床応用にむけ大きな注目を浴びている。本研究はゲノム編集技術を用いて超小型のマイクロミニブタを作出し、多くの研究機関でヒトに近い特徴を有するブタで幹細胞試験を可能にすることを目指した。超小型の免疫不全ブタが活用できれば、多くの研究機関で有用なモデル動物であるブタを用いた研究が可能となり、再生医療研究のみならず、がん研究など多様な研究分野に貢献可能であると思われる。

Research Products

• [Journal Article] Comparison of the effects of introducing the CRISPR/Cas9 system by microinjection and electroporation into porcine embryos at different stages (2021)
  • Author(s): Le AQ., Tanihara F., Wittayarat M., Namula Z., Sato Y., Lin Q., Takebayashi K., Hirata M., Otoi T.
  • Journal Title: BMC Research Notes Volume: 14 Issue: 1 Pages: 7-7
  • DOI: 10.1186/s13104-020-05412-8
  • NAID: 120007147670
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] One-Step Generation of Multiple Gene-Edited Pigs by Electroporation of the CRISPR/Cas9 System into Zygotes to Reduce Xenoantigen Biosynthesis (2021)
  • Author(s): Tanihara F., Hirata M., Nguyen TN., Sawamoto O., Kikuchi T., Otoi T.
  • Journal Title: International journal of molecular sciences Volume: 22 Issue: 5 Pages: 2249-2249
  • DOI: 10.3390/ijms22052249
  • NAID: 120007164926
  • Peer Reviewed / Open Access
• [Journal Article] Lipofection-Mediated Introduction of CRISPR/Cas9 System into Porcine Oocytes and Embryos (2021)
  • Author(s): Hirata M., Wittayarat M., Namula Z., Le AQ., Lin Q., Takebayashi K., Thongkittidilok C., Tanihara F., Otoi T.
  • Journal Title: Animals Volume: 11 Issue: 2 Pages: 578-578
  • DOI: 10.3390/ani11020578
  • NAID: 120007147677
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Introduction of a point mutation in the KRAS gene of in vitro fertilized porcine zygotes via electroporation of the CRISPR/Cas9 system with single-stranded oligodeoxynucleotides. (2021)
  • Author(s): Wittayarat M., Hirata M., Namula Z., Sato Y., Nguyen TN., Le AQ., Lin Q., Takebayashi K., Tanihara F., Otoi T.
  • Journal Title: Animal Science Journal, Volume: 92 Issue: 1
  • DOI: 10.1111/asj.13534
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] One-step genome editing of porcine zygotes through the electroporation of a CRISPR/Cas9 system with two guide RNAs (2020)
  • Author(s): Hirata M., Wittayarat M., Tanihara F., Sato Y., Namula Z., Le AQ., Lin Q., Takebayashi K., Otoi T.
  • Journal Title: In Vitro Cellular & Developmental Biology - Animal Volume: 56 Issue: 8 Pages: 614-621
  • DOI: 10.1007/s11626-020-00507-9
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Evaluation of multiple gene targeting in porcine embryos by the CRISPR/Cas9 system using electroporation (2020)
  • Author(s): Hirata M., Wittayarat M., Namula Z., Le AQ., Lin Q., Nguyen TN., Takebayashi K., Sato Y., Tanihara F., Otoi T.
  • Journal Title: Molecular Biology Reports Volume: 47 Issue: 7 Pages: 5073-5079
  • DOI: 10.1007/s11033-020-05576-3
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Efficient generation of GGTA1-deficient pigs by electroporation of the CRISPR/Cas9 system into in vitro-fertilized zygotes (2020)
  • Author(s): Tanihara F., Hirata M., Nguyen TN., Sawamoto O., Kikuchi T., Masako Doi., Otoi T.
  • Journal Title: BMC biotechnology Volume: 20 Issue: 1 Pages: 40-40
  • DOI: 10.1186/s12896-020-00638-7
  • NAID: 120007045051
  • Peer Reviewed / Open Access
• [Journal Article] Generation of CD163-edited pig via electroporation of the CRISPR/Cas9 system into porcine in vitro-fertilized zygotes (2020)
  • Author(s): Tanihara, F., Hirata, M., Nguyen, TN., Le, AQ., Wittayarat, M., Fahrudin, M., Hirano, T., Otoi, T.
  • Journal Title: Animal Biotechnology Volume: - Issue: 2 Pages: 147-154
  • DOI: 10.1080/10495398.2019.1668801
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] The relationship between embryonic development and the efficiency of target mutations in porcine endogenous retroviruses (PERVs) pol genes in porcine embryos (2019)
  • Author(s): Hirata, M., Wittayarat, M., Hirano, T., Nguyen, TN., Le, AQ., Namula, Z., Fahrudin, M., Tanihara, F., Otoi, T.
  • Journal Title: Animals Volume: 9 Issue: 9 Pages: 593-593
  • DOI: 10.3390/ani9090593
  • NAID: 120006877872
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Effects of concentration of CRISPR/Cas9 components on genetic mosaicism in cytoplasmic microinjected porcine embryos (2019)
  • Author(s): Tanihara F, Hirata M, Nguyen NT, LE QA, Hirano T, Otoi T.
  • Journal Title: Journal of Reproduction and Development Volume: 65 Issue: 3 Pages: 209-214
  • DOI: 10.1262/jrd.2018-116
  • NAID: 130007662977
  • ISSN: 0916-8818, 1348-4400
  • Peer Reviewed / Open Access
• [Journal Article] Relationship among ovarian follicular status, developmental competence of oocytes, and anti-Müllerian hormone levels: A comparative study in Japanese wild boar crossbred gilts and Large White gilts. (2019)
  • Author(s): Tanihara F, Hirata M, Iizuka S, Sairiki S, Nii M, Nguyen NT, Le QA, Hirano T, Otoi T.
  • Journal Title: Anim Sci J. Volume: 印刷中 Issue: 6 Pages: 712-718
  • DOI: 10.1111/asj.13200
  • Peer Reviewed
• [Presentation] ブタ体外受精卵におけるCRISPR/Cas9システムを用いた複数遺伝子の同時改変 (2019)
  • Author(s): 1.平田真樹、谷原史倫、Nhien Thi Nguyen、Quynh Anh Le、平野隆之、音井威重
  • Organizer: 第7回日本先進医工学ブタ研究会
• [Presentation] Effects of CRISPR/Cas9-mediated gene targeting of porcine endogenous retrovirus on the developmental competence of porcine embryos (2019)
  • Author(s): Hirata, M., Tanihara, F., Nguyen, TN., Le, AQ., Hirano, T., Otoi, T.
  • Organizer: The 15th Transgenic Technology Meeting (TT2019)
  • Int'l Joint Research
• [Presentation] ブタにおけるCrispr/Cas9システムを使用したゲノム編集効率化についての検討 (2018)
  • Author(s): 平田真樹、谷原史倫、Nhien Thi Nguyen、Zhao Namula、音井威重
  • Organizer: 日本ゲノム編集学会第3回大会
• [Presentation] ブタにおける成熟卵および1細胞期胚段階でのゲノム編集が胚盤胞の変異導入効率に及ぼす影響 (2018)
  • Author(s): 平田真樹、谷原史倫、Manita Wittayarat、Nhien Thi Nguyen、 Le Anh Quynh、Zhao Namula、新居雅宏、音井威重
  • Organizer: 第6回日本先進医工学ブタ研究会