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Modification of i-GONAD Suitable for Production of Genome-Edited C57BL/6 Inbred Mouse Strain
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) ribonucleoprotei...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226992/ https://www.ncbi.nlm.nih.gov/pubmed/32295056 http://dx.doi.org/10.3390/cells9040957 |
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author | Kobayashi, Yukari Aoshima, Takuya Ito, Ryota Shinmura, Ryota Ohtsuka, Masato Akasaka, Eri Sato, Masahiro Takabayashi, Shuji |
author_facet | Kobayashi, Yukari Aoshima, Takuya Ito, Ryota Shinmura, Ryota Ohtsuka, Masato Akasaka, Eri Sato, Masahiro Takabayashi, Shuji |
author_sort | Kobayashi, Yukari |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-7226992 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-72269922020-05-18 Modification of i-GONAD Suitable for Production of Genome-Edited C57BL/6 Inbred Mouse Strain Kobayashi, Yukari Aoshima, Takuya Ito, Ryota Shinmura, Ryota Ohtsuka, Masato Akasaka, Eri Sato, Masahiro Takabayashi, Shuji Cells Article 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. MDPI 2020-04-13 /pmc/articles/PMC7226992/ /pubmed/32295056 http://dx.doi.org/10.3390/cells9040957 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Kobayashi, Yukari Aoshima, Takuya Ito, Ryota Shinmura, Ryota Ohtsuka, Masato Akasaka, Eri Sato, Masahiro Takabayashi, Shuji Modification of i-GONAD Suitable for Production of Genome-Edited C57BL/6 Inbred Mouse Strain |
title | Modification of i-GONAD Suitable for Production of Genome-Edited C57BL/6 Inbred Mouse Strain |
title_full | Modification of i-GONAD Suitable for Production of Genome-Edited C57BL/6 Inbred Mouse Strain |
title_fullStr | Modification of i-GONAD Suitable for Production of Genome-Edited C57BL/6 Inbred Mouse Strain |
title_full_unstemmed | Modification of i-GONAD Suitable for Production of Genome-Edited C57BL/6 Inbred Mouse Strain |
title_short | Modification of i-GONAD Suitable for Production of Genome-Edited C57BL/6 Inbred Mouse Strain |
title_sort | modification of i-gonad suitable for production of genome-edited c57bl/6 inbred mouse strain |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226992/ https://www.ncbi.nlm.nih.gov/pubmed/32295056 http://dx.doi.org/10.3390/cells9040957 |
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