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ID 116340
著者
Nguyen, Nhien Thi Tokushima University
Sawamoto, Osamu Otsuka Pharmaceutical Factory
Kikuchi, Takeshi Otsuka Pharmaceutical Factory
キーワード
CRISPR/Cas9
GGTA1
CMAH
B4GALNT2
pig
in vitro fertilization
electroporation
資料タイプ
学術雑誌論文
抄録
Xenoantigens cause hyperacute rejection and limit the success of interspecific xenografts. Therefore, genes involved in xenoantigen biosynthesis, such as GGTA1, CMAH, and B4GALNT2, are key targets to improve the outcomes of xenotransplantation. In this study, we introduced a CRISPR/Cas9 system simultaneously targeting GGTA1, CMAH, and B4GALNT2 into in vitro-fertilized zygotes using electroporation for the one-step generation of multiple gene-edited pigs without xenoantigens. First, we optimized the combination of guide RNAs (gRNAs) targeting GGTA1 and CMAH with respect to gene editing efficiency in zygotes, and transferred electroporated embryos with the optimized gRNAs and Cas9 into recipient gilts. Next, we optimized the Cas9 protein concentration with respect to the gene editing efficiency when GGTA1, CMAH, and B4GALNT2 were targeted simultaneously, and generated gene-edited pigs using the optimized conditions. We achieved the one-step generation of GGTA1/CMAH double-edited pigs and GGTA1/CMAH/B4GALNT2 triple-edited pigs. Immunohistological analyses demonstrated the downregulation of xenoantigens; however, these multiple gene-edited pigs were genetic mosaics that failed to knock out some xenoantigens. Although mosaicism should be resolved, the electroporation technique could become a primary method for the one-step generation of multiple gene modifications in pigs aimed at improving pig-to-human xenotransplantation.
掲載誌名
International Journal of Molecular Sciences
ISSN
14220067
出版者
MDPI
22
5
開始ページ
2249
発行日
2021-02-24
権利情報
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
EDB ID
出版社版DOI
出版社版URL
フルテキストファイル
言語
eng
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出版社版
部局
生物資源系