Genetic humanized Mouse production by CRISPITT

2016 Fiscal Year Final Research Report

• Project/Area Number: 26870077
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Integrative animal science; Laboratory animal science
• Research Institution: University of Tsukuba
• Principal Investigator: MIZUNO Seiya 筑波大学, 医学医療系, 助教 (10633141)
• Research Collaborator: Sugiyama Fumihiro 筑波大学, 医学医療系, 教授 (90226481) ; Takahashi Takeshi 実験動物中央研究所, 免疫研究室, 室長 (80335215) ; Iijima Saori ; Dinh Thi Huong Tra
• Project Period (FY): 2014-04-01 – 2017-03-31
• Keywords: ゲノム編集

Outline of Final Research Achievements

In the past, the mice carried targeted gene mutation was produced by gene-targeting method using with embryonic stem cells. This way is reliable but long time and big money were required. Genome editing technology are changing the way to produce the gene targeted mouse. This make it possible to produce the mice carried desired mutation just by embryo-microinjection. Especially, CRISPR/Cas9 is most powerful tool. We can make not only knock-out but also knock-in mouse. To produce knock-in mouse by embryo-based genome editing, single guide RNA, Cas9, and donor DNA were co-microinjected. For short tag (e.g. FLAG, HA. V5) knock-in, single strand oligo DNA donor have beed used. In contrast, double strand DNA vectors were used for gene fragment knock-in.
In this study, we validated the several way to produce knock-in by embryo-based genome editing and succeed to produce more than 70 bp knock-in with synthesized single strand DNA and more than 7 kb fragment knock-in with plasmid donor.

Free Research Field

発生工学、実験動物