Molecular Characterization of Transgene Integration by Next-Generation Sequencing in Transgenic Cattle

As the number of transgenic livestock increases, reliable detection and molecular characterization of transgene integration sites and copy number are crucial not only for interpreting the relationship between the integration site and the specific phenotype but also for commercial and economic demand...

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Main Authors:	Zhang, Ran, Yin, Yinliang, Zhang, Yujun, Li, Kexin, Zhu, Hongxia, Gong, Qin, Wang, Jianwu, Hu, Xiaoxiang, Li, Ning
Format:	Online
Language:	English
Published:	Public Library of Science 2012
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3503979/

id	pubmed-3503979
recordtype	oai_dc
spelling	pubmed-35039792012-11-26 Molecular Characterization of Transgene Integration by Next-Generation Sequencing in Transgenic Cattle Zhang, Ran Yin, Yinliang Zhang, Yujun Li, Kexin Zhu, Hongxia Gong, Qin Wang, Jianwu Hu, Xiaoxiang Li, Ning Research Article As the number of transgenic livestock increases, reliable detection and molecular characterization of transgene integration sites and copy number are crucial not only for interpreting the relationship between the integration site and the specific phenotype but also for commercial and economic demands. However, the ability of conventional PCR techniques to detect incomplete and multiple integration events is limited, making it technically challenging to characterize transgenes. Next-generation sequencing has enabled cost-effective, routine and widespread high-throughput genomic analysis. Here, we demonstrate the use of next-generation sequencing to extensively characterize cattle harboring a 150-kb human lactoferrin transgene that was initially analyzed by chromosome walking without success. Using this approach, the sites upstream and downstream of the target gene integration site in the host genome were identified at the single nucleotide level. The sequencing result was verified by event-specific PCR for the integration sites and FISH for the chromosomal location. Sequencing depth analysis revealed that multiple copies of the incomplete target gene and the vector backbone were present in the host genome. Upon integration, complex recombination was also observed between the target gene and the vector backbone. These findings indicate that next-generation sequencing is a reliable and accurate approach for the molecular characterization of the transgene sequence, integration sites and copy number in transgenic species. Public Library of Science 2012-11-21 /pmc/articles/PMC3503979/ /pubmed/23185606 http://dx.doi.org/10.1371/journal.pone.0050348 Text en © 2012 Zhang et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Zhang, Ran Yin, Yinliang Zhang, Yujun Li, Kexin Zhu, Hongxia Gong, Qin Wang, Jianwu Hu, Xiaoxiang Li, Ning
spellingShingle	Zhang, Ran Yin, Yinliang Zhang, Yujun Li, Kexin Zhu, Hongxia Gong, Qin Wang, Jianwu Hu, Xiaoxiang Li, Ning Molecular Characterization of Transgene Integration by Next-Generation Sequencing in Transgenic Cattle
author_facet	Zhang, Ran Yin, Yinliang Zhang, Yujun Li, Kexin Zhu, Hongxia Gong, Qin Wang, Jianwu Hu, Xiaoxiang Li, Ning
author_sort	Zhang, Ran
title	Molecular Characterization of Transgene Integration by Next-Generation Sequencing in Transgenic Cattle
title_short	Molecular Characterization of Transgene Integration by Next-Generation Sequencing in Transgenic Cattle
title_full	Molecular Characterization of Transgene Integration by Next-Generation Sequencing in Transgenic Cattle
title_fullStr	Molecular Characterization of Transgene Integration by Next-Generation Sequencing in Transgenic Cattle
title_full_unstemmed	Molecular Characterization of Transgene Integration by Next-Generation Sequencing in Transgenic Cattle
title_sort	molecular characterization of transgene integration by next-generation sequencing in transgenic cattle
description	As the number of transgenic livestock increases, reliable detection and molecular characterization of transgene integration sites and copy number are crucial not only for interpreting the relationship between the integration site and the specific phenotype but also for commercial and economic demands. However, the ability of conventional PCR techniques to detect incomplete and multiple integration events is limited, making it technically challenging to characterize transgenes. Next-generation sequencing has enabled cost-effective, routine and widespread high-throughput genomic analysis. Here, we demonstrate the use of next-generation sequencing to extensively characterize cattle harboring a 150-kb human lactoferrin transgene that was initially analyzed by chromosome walking without success. Using this approach, the sites upstream and downstream of the target gene integration site in the host genome were identified at the single nucleotide level. The sequencing result was verified by event-specific PCR for the integration sites and FISH for the chromosomal location. Sequencing depth analysis revealed that multiple copies of the incomplete target gene and the vector backbone were present in the host genome. Upon integration, complex recombination was also observed between the target gene and the vector backbone. These findings indicate that next-generation sequencing is a reliable and accurate approach for the molecular characterization of the transgene sequence, integration sites and copy number in transgenic species.
publisher	Public Library of Science
publishDate	2012
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3503979/
_version_	1611934494527324160

Molecular Characterization of Transgene Integration by Next-Generation Sequencing in Transgenic Cattle

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