Development of gene targeting strategies for amphibian research using genome editing

• Project/Area Number: 15K06802
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Laboratory animal science
• Research Institution: Hiroshima University
• Principal Investigator: Suzuki Ken-ichi 広島大学, 理学研究科, 特任准教授 (90363043)
• Co-Investigator(Renkei-kenkyūsha): Hayashi Toshinori 鳥取大学, 医学部, 准教授 (60580925)
• Project Period (FY): 2015-04-01 – 2018-03-31
• Project Status: Completed (Fiscal Year 2017)
• Budget Amount: ¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000); Fiscal Year 2017: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000); Fiscal Year 2016: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000); Fiscal Year 2015: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
• Keywords: ネッタイツメガエル / アフリカツメガエル / イベリアトゲイモリ / ゲノム編集 / ノックイン / トランスジェニック / 両生類 / 再生 / 変態 / ノックアウト

Outline of Final Research Achievements

Following completion of the genome sequences of amphibians, gene targeting techniques have become increasingly important for the further development of amphibian research in the life sciences. Recent advances in genome editing using the CRISPR-Cas system now allow for reverse genetics in the amphibians, Xenopus laevis, Xenopus tropicalis, and Pleurodeles waltl. In order to refine these amphibians as model animals for life science research, we tried to develop a highly efficient and rapid workflow for gene knock-out, gene knock-in, conditional knock-out, and safe harbor transgenic systems.

Research Products

• [Int'l Joint Research] シンシナティ大学(米国)
• [Journal Article] Functional analysis of thyroid hormone receptor beta in Xenopus tropicalis founders using CRISPR-Cas (2017)
  • Author(s): Sakane Y, Iida M, Hasebe T, Fujii S, Buchholz DR, Ishizuya-Oka A, Yamamoto T, Suzuki KT
  • Journal Title: Biology Open Volume: 7 Pages: 1-9
  • DOI: 10.1242/bio.030338
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Reactivation of larval keratin gene (krt62.L) in blastema epithelium during Xenopus froglet limb regeneration. (2017)
  • Author(s): Satoh A, Mitogawa K, Saito N, Suzuki M, Suzuki KT, Ochi H, Makanae A.
  • Journal Title: Developmental Biology Volume: 432 Issue: 2 Pages: 265-272
  • DOI: 10.1016/j.ydbio.2017.10.015
  • Peer Reviewed / Open Access
• [Journal Article] A Simple Protocol for Loss-of-Function Analysis in Xenopus tropicalis founders using the CRISPR-Cas System (2017)
  • Author(s): Sakane Y, Suzuki K. T, Yamamoto T.
  • Journal Title: Methods Mol Biol Volume: inpress
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Clustered Xenopus keratin genes: A genomic, transcriptomic, and proteomic analysis. (2017)
  • Author(s): Suzuki KT, Suzuki M, Shigeta M, Fortriede JD, Takahashi S, Mawaribuchi S, Yamamoto T, Taira M, Fukui A.
  • Journal Title: Developmental Biology Volume: inpress
  • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Rapid and efficient analysis of gene function using CRISPR-Cas9 in Xenopus tropicalis founders (2016)
  • Author(s): Shigeta M, Sakane Y, Iida M, Suzuki M, Kashiwagi K, Kashiwagi A, Fujii S, Yamamoto K, Suzuki K
  • Journal Title: Genes to Cells Volume: in press
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Tracking dynamics of histone H3 lysine 9 acetylation in Xenopus laevis tail regeneration (2016)
  • Author(s): Suzuki M, Takagi C, Miura S, Sakane Y, Sakuma T, Sakamoto N, Endo T, Kamei Y, Kimura H, Yamamoto T, Ueno N, Suzuki K
  • Journal Title: Genes to Cells Volume: Vol.21 (4) Issue: 4 Pages: 358-369
  • DOI: 10.1111/gtc.12349
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the PITCH systems. (2016)
  • Author(s): Sakuma T, Nakade S, Sakane Y, Suzuki K, Yamamoto T.
  • Journal Title: Nature Protocols Volume: 11 Issue: 1 Pages: 118-113
  • DOI: 10.1038/nprot.2015.140
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] The Expression of TALEN before Fertilization Provides a Rapid Knock-Out Phenotype in Xenopus laevis Founder Embryos. (2015)
  • Author(s): Miyamoto K, Suzuki K, Suzuki M, Sakane Y, Sakuma T, Herberg S, Simeone A, Simpson D, Jullien J, Yamamoto T, Gurdon JB.
  • Journal Title: PLOS ONE Volume: 10(11) Issue: 11 Pages: e0142946-e0142946
  • DOI: 10.1371/journal.pone.0142946
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Presentation] ネッタイツメガエルにおけるゲノム編集 (2018)
  • Author(s): 鈴木賢一
  • Organizer: 平成29年度NBRPネッタイツメガエル技術講習会
  • Invited
• [Presentation] A streamlined workflow for rapid and efficient gene disruption by CRISPR-Cas9 in Xenopus tropicalis founders (2017)
  • Author(s): Shigeta M, Sakane Y, Iida M, Suzuki M, Kashiwagi K, Kashiwagi A, Fujii S, Yamamoto T and Suzuki K
  • Organizer: Joint Meeting of the German and Japanese Societies of Developmental Biologists
  • Place of Presentation: Kiel, Germany
  • Year and Date: 2017-03-17
  • Int'l Joint Research
• [Presentation] Establishment of CRISPR-Cas9-based genome editing in Xenopus tropicalis for analysis of disease-related genes (2017)
  • Author(s): Sakane Y, Kashiwagi K, Kashiwagi A, Yamamoto T, Suzuki KT
  • Organizer: The 8th Aquatic Animal Models of Human Disease Conference
  • Place of Presentation: Birmingham, AL, USA
  • Year and Date: 2017-01-09
  • Int'l Joint Research
• [Presentation] Clustered Xenopus keratin genes: A genomic, transcriptomic, and proteomic analysis (2016)
  • Author(s): Suzuki M, Suzuki KT, Shigeta M, Takahashi S, Mawaribuchi S, Yamamoto T, Taira M, Fukui A
  • Organizer: 第22回国際動物学会/第87回日本動物学会合同大会
  • Place of Presentation: Okinawa, Japan
  • Year and Date: 2016-11-14
  • Int'l Joint Research