Establishment of XYi-method for generation of multiple genes knock-in mice

• Project/Area Number: 26640050
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Laboratory animal science
• Research Institution: University of Tsukuba
• Principal Investigator: YAGAMI Ken-ichi 筑波大学, 医学医療系, 教授 (40166476)
• Co-Investigator(Kenkyū-buntansha): MIZUNO Seiya 筑波大学, 医学医療系, 助教 (10633141)
• Project Period (FY): 2014-04-01 – 2016-03-31
• Project Status: Completed (Fiscal Year 2015)
• Budget Amount: ¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000); Fiscal Year 2015: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000); Fiscal Year 2014: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
• Keywords: マウス / 複数遺伝子変異 / XYi法 / CRISPR/Cas9 / マウスES細胞 / Y染色体

Outline of Final Research Achievements

For producing multiple genes knock-in mouse, we tried to establish novel ES cell line and mouse strain carrying suicide and reporter genes on their Y chromosome.
First, we construct gene targeting vector including drug inducible suicide cassette (SRa-promoter and iCaspase3E gene) and fluorescence reporter cassette (EF1-promoter and EGFP gene). Because Knock-in into Y chromosome happen with very low frequency, we designed two CRISPR-nickase site in each 5’ and 3’homology arm region. These two CRISPR target DNA fragments were inserted in the px335 which carried U6-gRNA cassette and CBh-Cas9 D10A cassette. Then, these 3 vectors (one targeting vector and two CRISPR expression vectors) were electroplated into C57BL/6J mouse ES cells. We obtained one ES clone in which drug inducible suicide and reporter gene were expressed. The KI allele was confirmed by not only PCR but also Southern blotting.

Research Products

• [Journal Article] Peri-implantation lethality in mice carrying megabase-scale deletion on 5qc3.3 is caused by Exoc1 null mutation (2015)
  • Author(s): Seiya Mizuno, Kohei Takami, Yoko Daitoku, Yoko Tanimoto, Tra Thi Huong Dinh, Saori Mizuno-Iijima, Yoshikazu Hasegawa, Satoru Takahashi, Fumihiro Sugiyama, Ken-ichi Yagami
  • Journal Title: Scientific Reports Volume: 5 Issue: 1 Pages: 13632-13632
  • DOI: 10.1038/srep13632
  • NAID: 120007136187
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Presentation] CRISPR/Casを用いたマウスゲノム編集の実際 (2016)
  • Author(s): 水野 聖哉、高橋 智、杉山 文博、八神 健一
  • Organizer: 第121回日本解剖学会総会全国学術集会
  • Place of Presentation: ビッグパレット福島（郡山市）
  • Year and Date: 2016-03-28
  • Invited
• [Presentation] Applied Genome Editing with CRISPR/Cas9 Plasmid in Mice (2015)
  • Author(s): Seiya Mizuno, Kanako Kato, Saori Iijima, Yuko Hamada, Tra Dinh Thi Huong, Yoshikazu Hoshino, Yoko Tanimoto, Satoru Takahashi, Fumihiro Sugiyama, Ken-ichi Yagami
  • Organizer: トランスポゾン転移とゲノム編集技術に関する国際会議2015
  • Place of Presentation: 奈良県新公会堂（奈良市）
  • Year and Date: 2015-11-17
  • Int'l Joint Research
• [Presentation] Applied Genome Efiting with CRISPR/Cas9 Plasmid in Mice (2014)
  • Author(s): 水野聖哉・加藤花名子・飯島沙織・Dinh Thi Huong Tra・谷本陽子・大徳陽子・石田みゆき・高橋智・杉山文博・八神健一
  • Organizer: 28th International Mammalian Genome Conference
  • Place of Presentation: バーハーバー（USA）
  • Year and Date: 2014-10-26 – 2014-10-29
• [Presentation] Generation of gene-modified mice with CRISPR/Cas9 system (2014)
  • Author(s): 水野聖哉
  • Organizer: The 11th Annual Conference of Chinese Association for Laboratory Animal Science (CALAS)
  • Place of Presentation: 重慶（中国）
  • Year and Date: 2014-06-25 – 2014-06-27
  • Invited
• [Presentation] CRISPR/Cas9プラスミドを介したマウスゲノム編集の応用 (2014)
  • Author(s): 水野聖哉・加藤花名子・飯島沙織・Dinh Thi Huong Tra・谷本陽子・大徳陽子・石田みゆき・依馬正次・伊川正人・高橋智・杉山文博・八神健一
  • Organizer: 第61回日本実験動物学会総会
  • Place of Presentation: 札幌
  • Year and Date: 2014-05-15 – 2014-05-17
• [Remarks] 筑波大学実験動物学研究室
  • URL: http://www.md.tsukuba.ac.jp/basic-med/lab-animal/