期刊
CELLS
卷 9, 期 4, 页码 -出版社
MDPI
DOI: 10.3390/cells9040957
关键词
low-dose PMSG; genome editing; i-GONAD; C57BL/6J; in vivo electroporation; CRISPR/Cas9
类别
资金
- Ministry of Education, Science, Sports, and Culture, Japan [16K07087]
- Grants-in-Aid for Scientific Research [16K07087] Funding Source: KAKEN
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 Omega, 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 (approximate to 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 Omega 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.
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