Development of bicistronic cre driver mice using CRISPR/Cas9

• Project/Area Number: 15K14359
• 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: SUGIYAMA Fumihiro 筑波大学, 医学医療系, 教授 (90226481)
• Co-Investigator(Kenkyū-buntansha): 水野 聖哉 筑波大学, 医学医療系, 助教 (10633141)
• Project Period (FY): 2015-04-01 – 2017-03-31
• Project Status: Completed (Fiscal Year 2016)
• Budget Amount: ¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000); 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: Bicistronic / CRISPR/Cas9 / Mice / バイシストロニック / creドライバーマウス / マウス / Cre / Ins1 / bicistronic

Outline of Final Research Achievements

In this project, we generated novel cre driver mice for gene manipulation in pancreatic beta cells. Using the CRISPR/Cas9 system, stop codon sequences of Ins1 were targeted for insertion of cre, including 2A sequences. A founder of C57BL/6J-Ins1(em1 (cre) Utr) strain was produced from an oocyte injected with pX330 containing the sequences encoding gRNA and Cas9 and a DNA donor plasmid carrying 2A-cre. (R26GRR x C57BL/6J-Ins1(em1 (cre) Utr)) F1 mice were histologically characterized for cre-loxP recombination in the embryonic and adult stages; cre-loxP recombination was observed in all pancreatic islets examined in which almost all insulin-positive cells showed tdsRed fluorescence, suggesting beta cell-specific recombination. Taken together, these observations indicated that C57BL/6J-Ins1(em1 (cre) Utr) is useful for studies of glucose metabolism and the strategy of bicistronic cre knock-in using the CRISPR/Cas9 system could be useful for production of cre driver mice.

Research Products

• [Int'l Joint Research] Suez Canal University(Egypt)
• [Int'l Joint Research] Suez Canal University(Egypt)
• [Journal Article] Generation of CRISPR/Cas9-mediated bicistronic knock-in <i>ins1-cre</i> driver mice (2016)
  • Author(s): Yoshikazu Hasegawa, Yoshikazu Hoshino, Abdelaziz E. Ibrahim, Kanako Kato, Yoko Daitoku1, Yoko Tanimoto, Yoshihisa Ikeda, Hisashi Oishi, Satoru Takahashi, Atsushi Yoshiki, Ken-ichi Yagami, Hiroyoshi Iseki, Seiya Mizuno, Fumihiro Sugiyama
  • Journal Title: Experimental Animals Volume: 65 Issue: 3 Pages: 319-327
  • DOI: 10.1538/expanim.16-0016
  • NAID: 130006882371
  • ISSN: 0007-5124, 1341-1357, 1881-7122
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Presentation] CRISPR/Cas9を用いた膵β細胞特異的にCre酵素を発現するバイシストロニックCreドライバーマウスの開発 (2016)
  • Author(s): 長谷川賀一、星野貴一、アブデラジーイブラヒム、加藤花名子、大徳陽子、谷本陽子、池田祥久、大石久史、高橋智、八神健一、伊関大敬、水野聖哉、杉山文博
  • 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）
  • Year and Date: 2016-05-18