Production of pigs for safe xenotransplantation by controlling infectious porcine endogenous retrovirus (PERV)

• Project/Area Number: 17K19325
• Research Category: Grant-in-Aid for Challenging Research (Exploratory)
• Allocation Type: Multi-year Fund
• Research Field: Veterinary medical science, Animal science, and related fields
• Research Institution: The University of Tokushima
• Principal Investigator: OTOI Takeshige 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)
• Co-Investigator(Kenkyū-buntansha): 竹本 龍也 徳島大学, 先端酵素学研究所(オープンイノベ), 教授 (30443899) ; 谷原 史倫 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), 特任助教 (90754680)
• Project Period (FY): 2017-06-30 – 2019-03-31
• Project Status: Completed (Fiscal Year 2018)
• Budget Amount: ¥6,370,000 (Direct Cost: ¥4,900,000、Indirect Cost: ¥1,470,000); Fiscal Year 2018: ¥2,990,000 (Direct Cost: ¥2,300,000、Indirect Cost: ¥690,000); Fiscal Year 2017: ¥3,380,000 (Direct Cost: ¥2,600,000、Indirect Cost: ¥780,000)
• Keywords: 内在性レトロウイルス / ゲノム編集 / ブタ / 異種移植 / CRISPR/Cas９ / 代替臓器 / 受精卵

Outline of Final Research Achievements

The transmission of porcine endogenous retrovirus (PERV) to humans is a potential risk after xenotransplantation of porcine organs. We investigated the relationship between the embryonic development and the efficiency of target mutations in the PERV gene on porcine zygotes, to produce PERV-free pigs using the genome editing by electroporation of Cas9 protein (GEEP) system. The present results indicate that the developmental competence of zygotes may have been affected by the modification of PERV genes.

Academic Significance and Societal Importance of the Research Achievements

本研究の特色は、クローン技術を用いないで体外受精卵からPERV遺伝子をノックアウトすることである。使用したGEEP法は、妊娠率、産子作製率が格段に高いことから、今後の遺伝子改変ブタ作製の手法を一変させる可能性がある。本研究成果において、PERV遺伝子を含め複数の候補遺伝子をノックアウトできることを明らかにしたことから、移植用代替臓器の提供動物として安全なブタが得られることが期待される。

Research Products

• [Int'l Joint Research] 広東海洋大学農学部(中国)
• [Int'l Joint Research] プリンス・オブ・ソンクラー大学獣医学部(タイ)
• [Int'l Joint Research] ベトナム国立農業大学獣医学部(ベトナム)
• [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
• [Journal Article] Effects of Tris (hydroxymethyl) aminomethane on the quality of frozen-thawed boar spermatozoa. (2019)
  • Author(s): Namula Z, Tanihara F, Wittayarat M, Hirata M, Nguyen NT, Hirano T, Le QA, Nii M, Otoi T.
  • Journal Title: Acta Vet Hung. Volume: 67 Issue: 1 Pages: 106-114
  • DOI: 10.1556/004.2019.012
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Hypothermic storage of porcine zygotes in serum supplemented with chlorogenic acid. (2019)
  • Author(s): Thi Nguyen N, Hirata M, Tanihara F, Hirano T, Le QA, Nii M, Otoi T.
  • Journal Title: Reprod Domest Anim. Volume: 54 Issue: 5 Pages: 750-755
  • DOI: 10.1111/rda.13417
  • Peer Reviewed / Open Access
• [Journal Article] Genome mutation after introduction of the gene editing by electroporation of Cas9 protein (GEEP) system in matured oocytes and putative zygotes. (2019)
  • Author(s): Hirata M, Tanihara F, Wittayarat M, Hirano T, Nguyen NT, Le QA, Namula Z, Nii M, Otoi T.
  • Journal Title: In Vitro Cell Dev Biol Anim. Volume: 55 Issue: 4 Pages: 237-242
  • DOI: 10.1007/s11626-019-00338-3
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Effects of chlorogenic acid and caffeic acid on the quality of frozen-thawed boar sperm. (2018)
  • Author(s): Namula Z, Hirata M, Wittayarat M, Tanihara F, Thi Nguyen N, Hirano T, Nii M, Otoi T.
  • Journal Title: Reprod Domest Anim. Volume: 53 Issue: 6 Pages: 1600-1604
  • DOI: 10.1111/rda.13288
  • NAID: 120007099363
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Effects of chlorogenic acid (CGA) supplementation during in vitro maturation culture on the development and quality of porcine embryos with electroporation treatment after in vitro fertilization. (2018)
  • Author(s): Nguyen TV, Wittayarat M, Do LTK, Nguyen TV, Nii M, Namula Z, Kunihara T, Tanihara F, Hirata M, Otoi T.
  • Journal Title: Anim Sci J. Volume: 89 Issue: 8 Pages: 1207-1213
  • DOI: 10.1111/asj.13049
  • NAID: 120007099365
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Effects of Antifreeze Protein Supplementation on the Development of Porcine Morulae Stored at Hypothermic Temperatures. (2018)
  • Author(s): Nguyen TV, Tanihara F, Hirata M, Hirano T, Nishio K, Kim Do LT, Nguyen TV, Nii M, Otoi T.
  • Journal Title: Cryo Letters. Volume: 39 Pages: 131-136
  • Peer Reviewed / Int'l Joint Research
• [Presentation] ブタ体外受精卵におけるCrispr/Cas9システムを使用したゲノム編集の効率 (2018)
  • Author(s): 平田 真樹, 谷原 史倫, NGUYEN NHIEN THI, Namula Zhao, 音井 威重
  • Organizer: 日本ゲノム編集学会 第3回大会
• [Presentation] ブタ内在性レトロウイルス遺伝子を標的としたゲノム編集が胚発育能に及ぼす影響 (2018)
  • Author(s): 谷原 史倫, 平田 真樹, NGUYEN NHIEN THI, 平野 隆之, 音井 威重
  • Organizer: 日本ゲノム編集学会第3回大会
• [Presentation] Concentration of CRISPR/Cas9 components effects on genetic mosaicism of cytoplasmic microinjected porcine embryos (2018)
  • Author(s): Le Anh Quynh, Fuminori Tanihara, Maki Hirata, Nguyen Thi Nhien, Takayuki Hirano and Takeshige Otoi
  • Organizer: 第6回日本先進医工学ブタ研究会
• [Presentation] Hypothermic storage of porcine zygotes in serum supplemented with chlorogenic acid (2018)
  • Author(s): Nguyen Thi Nhien, Fuminori Tanihara, Maki Hirata, Takayuki Hirano, Le Anh Quynh, 新居 雅宏 and Takeshige Otoi
  • Organizer: 第6回日本先進医工学ブタ研究会
• [Presentation] ブタにおける受精前後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, 平野 隆之, NGUYEN NHIEN THI, LE ANH QUYNH, 新居 雅宏, 音井 威重
  • Organizer: 第6回日本先進医工学ブタ研究会
• [Presentation] ゲノム編集によるTP53遺伝子改変ブタの作製と表現型の解析 (2018)
  • Author(s): 谷原 史倫, 平田 真樹, NGUYEN NHIEN THI, LE ANH QUYNH, 平野 隆之, 竹本 龍也, 中井 美智子, 淵本 大一郎, 音井 威重
  • Organizer: 第6回日本先進医工学ブタ研究会
• [Presentation] Modification of SALL1 gene via CRISPR/Cas9-mediated gene editing introduced into porcine zygotes by electroporation (2018)
  • Author(s): Fuminori Tanihara, Tatsuya Takemoto, Maki Hirata, N Nguyen Thi, Toshiki Kunihara, R Nishinakamura and Takeshige Otoi
  • Organizer: The 3rd International symposium on Stem Cell Traits and Developmental Sys
  • Int'l Joint Research / Invited
• [Presentation] Effects of voltage strength on development and quality of electroporated porcine embryos. (2017)
  • Author(s): Nishio, K., Tanihara, F., Nguyen, T.V., Kunihara,T. and Otoi, T.
  • Organizer: 2017 Transgenic Technology Meeting
  • Int'l Joint Research
• [Remarks] 徳島大学・教育研究者総覧・音井・研究活動
  • URL: http://pub2.db.tokushima-u.ac.jp/ERD/person/292974/work-ja.html