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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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Autores principales: Zhang, Ran, Yin, Yinliang, Zhang, Yujun, Li, Kexin, Zhu, Hongxia, Gong, Qin, Wang, Jianwu, Hu, Xiaoxiang, Li, Ning
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3503979/
https://www.ncbi.nlm.nih.gov/pubmed/23185606
http://dx.doi.org/10.1371/journal.pone.0050348
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author Zhang, Ran
Yin, Yinliang
Zhang, Yujun
Li, Kexin
Zhu, Hongxia
Gong, Qin
Wang, Jianwu
Hu, Xiaoxiang
Li, Ning
author_facet Zhang, Ran
Yin, Yinliang
Zhang, Yujun
Li, Kexin
Zhu, Hongxia
Gong, Qin
Wang, Jianwu
Hu, Xiaoxiang
Li, Ning
author_sort Zhang, Ran
collection PubMed
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.
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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 PLoS One 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.
spellingShingle Research Article
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
title 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_short 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
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3503979/
https://www.ncbi.nlm.nih.gov/pubmed/23185606
http://dx.doi.org/10.1371/journal.pone.0050348
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