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Screen and Verification for Transgene Integration Sites in Pigs

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 g...

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Autores principales: Ma, Linyuan, Wang, Yuzhe, Wang, Haitao, Hu, Yiqing, Chen, Jingyao, Tan, Tan, Hu, Man, Liu, Xiaojuan, Zhang, Ran, Xing, Yiming, Zhao, Yiqiang, Hu, Xiaoxiang, Li, Ning
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5943519/
https://www.ncbi.nlm.nih.gov/pubmed/29743638
http://dx.doi.org/10.1038/s41598-018-24481-1
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author Ma, Linyuan
Wang, Yuzhe
Wang, Haitao
Hu, Yiqing
Chen, Jingyao
Tan, Tan
Hu, Man
Liu, Xiaojuan
Zhang, Ran
Xing, Yiming
Zhao, Yiqiang
Hu, Xiaoxiang
Li, Ning
author_facet Ma, Linyuan
Wang, Yuzhe
Wang, Haitao
Hu, Yiqing
Chen, Jingyao
Tan, Tan
Hu, Man
Liu, Xiaojuan
Zhang, Ran
Xing, Yiming
Zhao, Yiqiang
Hu, Xiaoxiang
Li, Ning
author_sort Ma, Linyuan
collection PubMed
description 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.
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spelling pubmed-59435192018-05-14 Screen and Verification for Transgene Integration Sites in Pigs Ma, Linyuan Wang, Yuzhe Wang, Haitao Hu, Yiqing Chen, Jingyao Tan, Tan Hu, Man Liu, Xiaojuan Zhang, Ran Xing, Yiming Zhao, Yiqiang Hu, Xiaoxiang Li, Ning Sci Rep Article 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. Nature Publishing Group UK 2018-05-09 /pmc/articles/PMC5943519/ /pubmed/29743638 http://dx.doi.org/10.1038/s41598-018-24481-1 Text en © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Ma, Linyuan
Wang, Yuzhe
Wang, Haitao
Hu, Yiqing
Chen, Jingyao
Tan, Tan
Hu, Man
Liu, Xiaojuan
Zhang, Ran
Xing, Yiming
Zhao, Yiqiang
Hu, Xiaoxiang
Li, Ning
Screen and Verification for Transgene Integration Sites in Pigs
title 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_short Screen and Verification for Transgene Integration Sites in Pigs
title_sort screen and verification for transgene integration sites in pigs
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5943519/
https://www.ncbi.nlm.nih.gov/pubmed/29743638
http://dx.doi.org/10.1038/s41598-018-24481-1
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