title: 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.
authors.affiliation: State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing, China.
authors.forename: Ran
authors.initials: R
authors.lastname: Zhang
authors.forename: Yinliang
authors.initials: Y
authors.lastname: Yin
authors.forename: Yujun
authors.initials: Y
authors.lastname: Zhang
authors.forename: Kexin
authors.initials: K
authors.lastname: Li
authors.forename: Hongxia
authors.initials: H
authors.lastname: Zhu
authors.forename: Qin
authors.initials: Q
authors.lastname: Gong
authors.forename: Jianwu
authors.initials: J
authors.lastname: Wang
authors.forename: Xiaoxiang
authors.initials: X
authors.lastname: Hu
authors.forename: Ning
authors.initials: N
authors.lastname: Li
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D002417:Cattle / Q000235:genetics*; D018628:Gene Dosage; D005822:Genetic Vectors; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007781:Lactoferrin / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D019076:Transgenes
chemical_list: D007781:Lactoferrin