Screen and Verification for Transgene Integration Sites in Pigs

Abstract Efficient transgene expression in recipient cells constitutes the primary step in gene therapy. However, random integration in host genome comprises too many uncertainties. Our study presents a strategy combining bioinformatics and functional verification to find transgene integration sites...

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Autores principales:	Linyuan Ma, Yuzhe Wang, Haitao Wang, Yiqing Hu, Jingyao Chen, Tan Tan, Man Hu, Xiaojuan Liu, Ran Zhang, Yiming Xing, Yiqiang Zhao, Xiaoxiang Hu, Ning Li
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2018
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/e4e5e16e3fb34d098b20761d1fcbc221
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spelling	oai:doaj.org-article:e4e5e16e3fb34d098b20761d1fcbc2212021-12-02T11:40:46ZScreen and Verification for Transgene Integration Sites in Pigs10.1038/s41598-018-24481-12045-2322https://doaj.org/article/e4e5e16e3fb34d098b20761d1fcbc2212018-05-01T00:00:00Zhttps://doi.org/10.1038/s41598-018-24481-1https://doaj.org/toc/2045-2322Abstract Efficient transgene expression in recipient cells constitutes the primary step in gene therapy. However, random integration in host genome comprises too many uncertainties. Our study presents a strategy combining bioinformatics and functional verification to find transgene integration sites in pig genome. Using an in silico approach, we screen out two candidate sites, namely, Pifs302 and Pifs501, located in actively transcribed intergenic regions with low nucleosome formation potential and without potential non-coding RNAs. After CRISPR/Cas9-mediated site-specific integration on Pifs501, we detected high EGFP expression in different pig cell types and ubiquitous EGFP expression in diverse tissues of transgenic pigs without adversely affecting 600 kb neighboring gene expression. Promoters integrated on Pifs501 exhibit hypomethylated modification, which suggest a permissive epigenetic status of this locus. We establish a versatile master cell line on Pifs501, which allows us to achieve site-specific exchange of EGFP to Follistatin with Cre/loxP system conveniently. Through in vitro and in vivo functional assays, we demonstrate the effectiveness of this screening method, and take Pifs501 as a potential site for transgene insertion in pigs. We anticipate that Pifs501 will have useful applications in pig genome engineering, though the identification of genomic safe harbor should over long-term various functional studies.Linyuan MaYuzhe WangHaitao WangYiqing HuJingyao ChenTan TanMan HuXiaojuan LiuRan ZhangYiming XingYiqiang ZhaoXiaoxiang HuNing LiNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 8, Iss 1, Pp 1-11 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Linyuan Ma Yuzhe Wang Haitao Wang Yiqing Hu Jingyao Chen Tan Tan Man Hu Xiaojuan Liu Ran Zhang Yiming Xing Yiqiang Zhao Xiaoxiang Hu Ning Li Screen and Verification for Transgene Integration Sites in Pigs
description	Abstract Efficient transgene expression in recipient cells constitutes the primary step in gene therapy. However, random integration in host genome comprises too many uncertainties. Our study presents a strategy combining bioinformatics and functional verification to find transgene integration sites in pig genome. Using an in silico approach, we screen out two candidate sites, namely, Pifs302 and Pifs501, located in actively transcribed intergenic regions with low nucleosome formation potential and without potential non-coding RNAs. After CRISPR/Cas9-mediated site-specific integration on Pifs501, we detected high EGFP expression in different pig cell types and ubiquitous EGFP expression in diverse tissues of transgenic pigs without adversely affecting 600 kb neighboring gene expression. Promoters integrated on Pifs501 exhibit hypomethylated modification, which suggest a permissive epigenetic status of this locus. We establish a versatile master cell line on Pifs501, which allows us to achieve site-specific exchange of EGFP to Follistatin with Cre/loxP system conveniently. Through in vitro and in vivo functional assays, we demonstrate the effectiveness of this screening method, and take Pifs501 as a potential site for transgene insertion in pigs. We anticipate that Pifs501 will have useful applications in pig genome engineering, though the identification of genomic safe harbor should over long-term various functional studies.
format	article
author	Linyuan Ma Yuzhe Wang Haitao Wang Yiqing Hu Jingyao Chen Tan Tan Man Hu Xiaojuan Liu Ran Zhang Yiming Xing Yiqiang Zhao Xiaoxiang Hu Ning Li
author_facet	Linyuan Ma Yuzhe Wang Haitao Wang Yiqing Hu Jingyao Chen Tan Tan Man Hu Xiaojuan Liu Ran Zhang Yiming Xing Yiqiang Zhao Xiaoxiang Hu Ning Li
author_sort	Linyuan Ma
title	Screen and Verification for Transgene Integration Sites in Pigs
title_short	Screen and Verification for Transgene Integration Sites in Pigs
title_full	Screen and Verification for Transgene Integration Sites in Pigs
title_fullStr	Screen and Verification for Transgene Integration Sites in Pigs
title_full_unstemmed	Screen and Verification for Transgene Integration Sites in Pigs
title_sort	screen and verification for transgene integration sites in pigs
publisher	Nature Portfolio
publishDate	2018
url	https://doaj.org/article/e4e5e16e3fb34d098b20761d1fcbc221
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_version_	1718395553476247552

Screen and Verification for Transgene Integration Sites in Pigs

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