Modification of i-GONAD Suitable for Production of Genome-Edited C57BL/6 Inbred Mouse Strain

Cells

Masato Ohtsuka¹  Ryota Shinmura²  Takuya Aoshima²  Shuji Takabayashi²  Yukari Kobayashi²  Ryota Ito²  Eri Akasaka³  Masahiro Sato³ 

[1] Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Isehara, Kanagawa 259-1193, Japan;Laboratory Animal Facilities & Services, Preeminent Medical Photonics Education & Research Center, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu, Shizuoka 431-3192, Japan;Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Kagoshima 890-8544, Japan;

关键词: low-dose PMSG;    genome editing;    i-GONAD;    C57BL/6J;    in vivo electroporation;    CRISPR/Cas9;   

DOI  :  10.3390/cells9040957

来源: DOAJ

【 摘 要 】

Improved genome editing via oviductal nucleic acid delivery (i-GONAD) is a novel method for producing genome-edited mice in the absence of ex vivo handling of zygotes. i-GONAD involves the intraoviductal injection of clustered regularly interspaced short palindromic repeats (CRISPR) ribonucleoproteins via the oviductal wall of pregnant females at 0.7 days post-coitum, followed by in vivo electroporation (EP). Unlike outbred Institute of Cancer Research (ICR) and hybrid mouse strains, genome editing of the most widely used C57BL/6J (B6) strain with i-GONAD has been considered difficult but, recently, setting a constant current of 100 mA upon EP enabled successful i-GONAD in this strain. Unfortunately, the most widely used electroporators employ a constant voltage, and thus we explored conditions allowing the generation of a 100 mA current using two electroporators: NEPA21 (Nepa Gene Co., Ltd.) and GEB15 (BEX Co., Ltd.). When the current and resistance were set to 40 V and 350–400 Ω, respectively, the current was fixed to 100 mA. Another problem in using B6 mice for i-GONAD is the difficulty in obtaining pregnant B6 females consistently because estrous females often fail to be found. A single intraperitoneal injection of low-dose pregnant mare’s serum gonadotrophin (PMSG) led to synchronization of the estrous cycle of these mice. Consequently, approximately 51% of B6 females had plugs upon mating with males 2 days after PMSG administration, which contrasts with the case (≈26%) when B6 females were subjected to natural mating. i-GONAD performed on PMSG-treated pregnant B6 females under conditions of average resistance of 367 Ω and average voltage of 116 mA resulted in the production of pregnant females at a rate of 56% (5/9 mice), from which 23 fetuses were successfully delivered. Nine (39%) of these fetuses exhibited successful genome editing at the target locus.

【 授权许可】

Unknown