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.

Molecular Characterization of Transgenic Rice Producing Resveratrol

“…The multiple or incomplete integration events pose technical challenges for characterization of transgenes using PCR-based chromosome-walking techniques (Tonooka et al 2009;Zhang et al 2012). In this study, we were confronted with a severe challenge for conventional PCR amplification due to the poly G region of the ubiquitin promoter.…”

Copy number and integration sites in growth hormone transgenic goats

“…Several reports demonstrated that integration sites influence transgene expression levels (Grosveld et al, 1987;Williams et al, 2008), while in some case, integration positions have no effect on transgene expression (Wang et al, 2010). However, the inserted DNA may cause rearrangement of endogenous genes (Le Saux et al, 2010;Zhang et al, 2012) and result in genome variation. Several PCR-based methods have been described to precisely determine the integration site of exogenous DNA into native chromosomes, including inverse PCR , ligation-mediated PCR (Yuanxin et al, 2003), and TAIL-PCR (Yan et al, 2010(Yan et al, , 2013.…”

“…Somatic cell nuclear transfer (SCNT) is a conventional method for gene transfer into livestock, in which foreign DNA fragments often randomly integrate into the genome of donor cell, resulting in tandem repeats encompassing several copies of exogenous genes. Additionally, random integration of exogenous gene can often cause deletions or rearrangements in the genome (Le Provost et al, 2010;Zhang et al, 2012). When new transgenic animals are produced, an essential step is to determine the copy number and insertion sites of the inserted genes in the transgenic animals.…”

Illuminating the Role of Next-Generation Sequencing Approaches in Transgenesis

“…In one of the recent studies, the authors successfully demonstrated the utilization of next-generation sequencing technology to characterize cattle harboring a 150 kb Bacterial artificial chromosome (BAC) that expresses human lactoferrin [1]. The author's initial attempt to characterize it with the conventional chromosome walking was met with no success [1].…”

“…The author's initial attempt to characterize it with the conventional chromosome walking was met with no success [1].…”