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

Yukari Kobayashi,Takuya Aoshima,Masahiro Sato,Ryota Ito,Shuji Takabayashi,Ryota Shinmura,Masato Ohtsuka,Eri Akasaka

doi:10.3390/cells9040957

Yukari Kobayashi, Takuya Aoshima + Show 6 more

Open Access

https://doi.org/10.3390/cells9040957

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Abstract

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.

Highlights

Clustered regularly interspaced short palindromic repeats (CRISPR)/caspase-9 (Cas9)(CRISPR/Cas9) gene editing technology has been widely employed for the rapid generation of genetically modified (GM) animals, due to its simplicity, versatility, and efficiency [1,2]
We examined whether the administration of a single IP injection of low-dose pregnant mare’s serum gonadotrophin (PMSG) is effective for synchronizing the estrous cycle in B6 females
We explored optimal electric conditions that allow the i-genome editing via oviductal nucleic acid delivery (GONAD)-based production of the genome-edited B6 strain, as well as other inbred strains such as BALB/c when a universal electroporator such as NEAP21 and GEB15 is employed

Summary

Introduction

Clustered regularly interspaced short palindromic repeats (CRISPR)/caspase-9 (Cas9)(CRISPR/Cas9) gene editing technology has been widely employed for the rapid generation of genetically modified (GM) animals, due to its simplicity, versatility, and efficiency [1,2]. We developed a new method, called genome editing via oviductal nucleic acid delivery (GONAD), which was subsequently renamed “improved GONAD (i-GONAD)”, for the production of genome-edited mice [16,17,18], rats [19,20], and hamsters [21] This technology is based on the injection of a solution (1–1.5 μL) containing genome editing reagents into the lumen of an oviduct via the oviductal wall of pregnant female animals at the late zygote to two-cell stage following in vivo EP of the entire oviduct using tweezer-type electrodes under a dissecting microscope [22,23]. GONAD/i-GONAD appears to be more convenient and simpler than the methods that are based on the ex vivo handling of embryos

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