2020年4月13日
Modification of i-GONAD Suitable for Production of Genome-Edited C57BL/6 Inbred Mouse Strain.
Cells
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- 巻
- 9
- 号
- 4
- 記述言語
- 英語
- 掲載種別
- 研究論文(学術雑誌)
- DOI
- 10.3390/cells9040957
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.
- リンク情報
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- DOI
- https://doi.org/10.3390/cells9040957
- PubMed
- https://www.ncbi.nlm.nih.gov/pubmed/32295056
- PubMed Central
- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226992
- Scopus
- https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85083477026&origin=inward 本文へのリンクあり
- Scopus Citedby
- https://www.scopus.com/inward/citedby.uri?partnerID=HzOxMe3b&scp=85083477026&origin=inward
- ID情報
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- DOI : 10.3390/cells9040957
- eISSN : 2073-4409
- PubMed ID : 32295056
- PubMed Central 記事ID : PMC7226992
- SCOPUS ID : 85083477026