Dataset Information

Molecular characterization of transgene integration by next-generation sequencing in transgenic cattle.

ABSTRACT: 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.

SUBMITTER: Zhang R

PROVIDER: S-EPMC3503979 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Molecular characterization of transgene integration by next-generation sequencing in transgenic cattle.

Zhang Ran R Yin Yinliang Y Zhang Yujun Y Li Kexin K Zhu Hongxia H Gong Qin Q Wang Jianwu J Hu Xiaoxiang X Li Ning N

PloS one 20121121 11

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 se ...[more]

PMID: 23185606

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Project description:BackgroundThe molecular characterization information of T-DNA integration is not only required by public risk assessors and regulators, but is also closely related to the expression of exogenous and endogenous genes. At present, with the development of sequencing technology, whole-genome resequencing has become an attractive approach to identify unknown genetically modified events and characterise T-DNA integration events.ResultsIn this study, we performed genome resequencing of Pb29, a transgenic high-resistance poplar 741 line that has been commercialized, using next-generation and Nanopore sequencing. The results revealed that there are two T-DNA insertion sites, located at 9,283,905-9,283,937 bp on chromosome 3 (Chr03) and 10,868,777-10,868,803 bp on Chr10. The accuracy of the T-DNA insertion locations and directions was verified using polymerase chain reaction amplification. Through sequence alignment, different degrees of base deletions were detected on the T-DNA left and right border sequences, and in the flanking sequences of the insertion sites. An unknown fragment was inserted between the Chr03 insertion site and the right flanking sequence, but the Pb29 genome did not undergo chromosomal rearrangement. It is worth noting that we did not detect the API gene in the Pb29 genome, indicating that Pb29 is a transgenic line containing only the BtCry1AC gene. On Chr03, the insertion of T-DNA disrupted a gene encoding TAF12 protein, but the transcriptional abundance of this gene did not change significantly in the leaves of Pb29. Additionally, except for the gene located closest to the T-DNA integration site, the expression levels of four other neighboring genes did not change significantly in the leaves of Pb29.ConclusionsThis study provides molecular characterization information of T-DNA integration in transgenic poplar 741 line Pb29, which contribute to safety supervision and further extensive commercial planting of transgenic poplar 741.

Dataset Information

Molecular characterization of transgene integration by next-generation sequencing in transgenic cattle.

Publications

Molecular characterization of transgene integration by next-generation sequencing in transgenic cattle.

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