Optimization of a CRISPR/Cas9-mediated Knock-in Strategy at the Porcine Rosa26 Locus in Porcine Foetal Fibroblasts

Abstract Genetically modified pigs have important roles in agriculture and biomedicine. However, genome-specific knock-in techniques in pigs are still in their infancy and optimal strategies have not been extensively investigated. In this study, we performed electroporation to introduce a targeting...

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Autores principales:	Zicong Xie, Daxin Pang, Kankan Wang, Mengjing Li, Nannan Guo, Hongming Yuan, Jianing Li, Xiaodong Zou, Huping Jiao, Hongsheng Ouyang, Zhanjun Li, Xiaochun Tang
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/e60be4d4d34e431daab183a5b1326ffc
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spelling	oai:doaj.org-article:e60be4d4d34e431daab183a5b1326ffc2021-12-02T11:40:14ZOptimization of a CRISPR/Cas9-mediated Knock-in Strategy at the Porcine Rosa26 Locus in Porcine Foetal Fibroblasts10.1038/s41598-017-02785-y2045-2322https://doaj.org/article/e60be4d4d34e431daab183a5b1326ffc2017-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-02785-yhttps://doaj.org/toc/2045-2322Abstract Genetically modified pigs have important roles in agriculture and biomedicine. However, genome-specific knock-in techniques in pigs are still in their infancy and optimal strategies have not been extensively investigated. In this study, we performed electroporation to introduce a targeting donor vector (a non-linearized vector that did not contain a promoter or selectable marker) into Porcine Foetal Fibroblasts (PFFs) along with a CRISPR/Cas9 vector. After optimization, the efficiency of the EGFP site-specific knock-in could reach up to 29.6% at the pRosa26 locus in PFFs. Next, we used the EGFP reporter PFFs to address two key conditions in the process of achieving transgenic pigs, the limiting dilution method and the strategy to evaluate the safety and feasibility of the knock-in locus. This study demonstrates that we establish an efficient procedures for the exogenous gene knock-in technique and creates a platform to efficiently generate promoter-less and selectable marker-free transgenic PFFs through the CRISPR/Cas9 system. This study should contribute to the generation of promoter-less and selectable marker-free transgenic pigs and it may provide insights into sophisticated site-specific genome engineering techniques for additional species.Zicong XieDaxin PangKankan WangMengjing LiNannan GuoHongming YuanJianing LiXiaodong ZouHuping JiaoHongsheng OuyangZhanjun LiXiaochun TangNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-12 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Zicong Xie Daxin Pang Kankan Wang Mengjing Li Nannan Guo Hongming Yuan Jianing Li Xiaodong Zou Huping Jiao Hongsheng Ouyang Zhanjun Li Xiaochun Tang Optimization of a CRISPR/Cas9-mediated Knock-in Strategy at the Porcine Rosa26 Locus in Porcine Foetal Fibroblasts
description	Abstract Genetically modified pigs have important roles in agriculture and biomedicine. However, genome-specific knock-in techniques in pigs are still in their infancy and optimal strategies have not been extensively investigated. In this study, we performed electroporation to introduce a targeting donor vector (a non-linearized vector that did not contain a promoter or selectable marker) into Porcine Foetal Fibroblasts (PFFs) along with a CRISPR/Cas9 vector. After optimization, the efficiency of the EGFP site-specific knock-in could reach up to 29.6% at the pRosa26 locus in PFFs. Next, we used the EGFP reporter PFFs to address two key conditions in the process of achieving transgenic pigs, the limiting dilution method and the strategy to evaluate the safety and feasibility of the knock-in locus. This study demonstrates that we establish an efficient procedures for the exogenous gene knock-in technique and creates a platform to efficiently generate promoter-less and selectable marker-free transgenic PFFs through the CRISPR/Cas9 system. This study should contribute to the generation of promoter-less and selectable marker-free transgenic pigs and it may provide insights into sophisticated site-specific genome engineering techniques for additional species.
format	article
author	Zicong Xie Daxin Pang Kankan Wang Mengjing Li Nannan Guo Hongming Yuan Jianing Li Xiaodong Zou Huping Jiao Hongsheng Ouyang Zhanjun Li Xiaochun Tang
author_facet	Zicong Xie Daxin Pang Kankan Wang Mengjing Li Nannan Guo Hongming Yuan Jianing Li Xiaodong Zou Huping Jiao Hongsheng Ouyang Zhanjun Li Xiaochun Tang
author_sort	Zicong Xie
title	Optimization of a CRISPR/Cas9-mediated Knock-in Strategy at the Porcine Rosa26 Locus in Porcine Foetal Fibroblasts
title_short	Optimization of a CRISPR/Cas9-mediated Knock-in Strategy at the Porcine Rosa26 Locus in Porcine Foetal Fibroblasts
title_full	Optimization of a CRISPR/Cas9-mediated Knock-in Strategy at the Porcine Rosa26 Locus in Porcine Foetal Fibroblasts
title_fullStr	Optimization of a CRISPR/Cas9-mediated Knock-in Strategy at the Porcine Rosa26 Locus in Porcine Foetal Fibroblasts
title_full_unstemmed	Optimization of a CRISPR/Cas9-mediated Knock-in Strategy at the Porcine Rosa26 Locus in Porcine Foetal Fibroblasts
title_sort	optimization of a crispr/cas9-mediated knock-in strategy at the porcine rosa26 locus in porcine foetal fibroblasts
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/e60be4d4d34e431daab183a5b1326ffc
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Optimization of a CRISPR/Cas9-mediated Knock-in Strategy at the Porcine Rosa26 Locus in Porcine Foetal Fibroblasts

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