2020
DOI: 10.3390/cells9040957
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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… Show more

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Cited by 13 publications
(13 citation statements)
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References 29 publications
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“…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.…”
Section: Strain-difference Can Affect the Efficiency Of I-gonad-media...mentioning
confidence: 85%
See 1 more Smart Citation
“…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.…”
Section: Strain-difference Can Affect the Efficiency Of I-gonad-media...mentioning
confidence: 85%
“…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%.…”
Section: Strain-difference Can Affect the Efficiency Of I-gonad-media...mentioning
confidence: 99%
“…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 ).…”
Section: Methodsmentioning
confidence: 99%
“…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: ±).…”
Section: Methodsmentioning
confidence: 99%
“…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.…”
Section: In Vivo Delivery Of Crispr/cas Systemsmentioning
confidence: 99%