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

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.

Recent Advances in In Vivo Genome Editing Targeting Mammalian Preimplantation Embryos

“…For example, it worked successful under relatively stringent electrical conditions (40 V/100-200 Ω/~300 mA) when random-bred mice (such as MCH(ICR) and B6C3F1, a hybrid between C3H/He and C57BL/6), but not C57BL/6 strain, were used. Under less stringent conditions (40 V/350-400 Ω/~100 mA), i-GONAD was successful in the inbred C57BL/6 strain [86,98,117]. These findings suggest the importance of selecting the appropriate EP conditions, particularly when different mouse strains are used for i-GONAD experiment.…”

“…Also, other electroporators (as exemplified by GEB15 (BEX Co., Ltd.)) employ a constant current. Kobayashi et al [98] explored the conditions allowing the generation of a 100 mA current in C57BL/6 mice using two electroporators, NEPA21 and GEB15. As a result, i-GONAD performed under conditions of average resistance of 367 Ω and average voltage of 116 mA resulted in the production of genome-edited fetuses with efficiency of 39%.…”

An efficient i-GONAD method for creating and maintaining lethal mutant mice using an inversion balancer identified from the C3H/HeJJcl strain

“…To synchronize the estrous cycle of female mice, 8–12-weeks-old female mice were injected intraperitoneally with 2.4 IU pregnant mare serum gonadotropin and mated with 8–24-weeks-old males 48 hours later, as previously described ( Kobayashi et al 2020 ). The presence of copulation plugs was confirmed the next morning via visual inspection, and plug-positive mice were subjected to i -GONAD experiments, as previously described ( Ohtsuka et al 2018 ; Gurumurthy et al 2019b ).…”

“…The CRISPR mixture (1 μl) was injected into the oviductal lumen upstream of the ampulla with a glass micropipette, which was made using a vertical capillary puller (NARISHIGE, Tokyo, Japan). Following injection of CRISPR solutions, the oviduct regions were grasped using tweezer electrodes (CUY652P2.5 × 4; Nepa Gene, Chiba, Japan), and electroporation was performed as previously described ( Kobayashi et al 2020 ) using a NEPA21 (Nepa Gene). The following parameters were used for electroporation: poring pulse (voltage: 40 V; pulse length: 5.0 ms; pulse interval: 50 ms; number of pulses: 3; decay rate: 10%; polarity: ±), transfer pulse (voltage: 10 V; pulse length: 50 ms; pulse interval: 50 ms; number of pulses: 3; decay rate: 40%; polarity: ±).…”

A most formidable arsenal: genetic technologies for building a better mouse

“…A similar protocol, CRISPR RNP electroporation of zygotes (CRISPR-EZ), also uses RNP and ssDNA, reporting successful HDR in 42% of live pups (Chen et al 2016). Alternatively, the improved genome-editing via oviductal nucleic acids delivery (i-GONAD) technique uses direct electroporation of the oviduct in pregnant females to induce CRISPR/Cas editing without ex vivo manipulation of zygotes (Takahashi et al 2015;Ohtsuka et al 2018;Kobayashi et al 2020;Sato et al 2020). These protocols highlight the strength of electroporation: high-throughput processing of embryos without requiring as much technical expertise as direct injection.…”