Identification of the Genomic Insertion Site of Pmel-1 TCR α and β Transgenes by Next-Generation Sequencing

Abstract

The pmel-1 T cell receptor transgenic mouse has been extensively employed as an ideal model system to study the mechanisms of tumor immunology, CD8+ T cell differentiation, autoimmunity and adoptive immunotherapy. The ‘zygosity’ of the transgene affects the transgene expression levels and may compromise optimal breeding scheme design. However, the integration sites for the pmel-1 mouse have remained uncharacterized. This is also true for many other commonly used transgenic mice created before the modern era of rapid and inexpensive next-generation sequencing. Here, we show that whole genome sequencing can be used to determine the exact pmel-1 genomic integration site, even with relatively ‘shallow’ (8X) coverage. The results were used to develop a validated polymerase chain reaction-based genotyping assay. For the first time, we provide a quick and convenient polymerase chain reaction method to determine the dosage of pmel-1 transgene for this freely and publically available mouse resource. We also demonstrate that next-generation sequencing provides a feasible approach for mapping foreign DNA integration sites, even when information of the original vector sequences is only partially known.

Identification of the Genomic Insertion Site of the Thyroid Peroxidase Promoter–Cre Recombinase Transgene Using a Novel, Efficient, Next-Generation DNA Sequencing Method

“…This method, however, requires designing a custom microarray for targeted sequence capture and enrichment. Shallow coverage whole genome sequencing has also been carried out in combination with searching for tandem duplications to help identify the transgenic insert, although this method would not be feasible for transgenic inserts without tandem repeats (11). The present method of combining PCR with next-generation sequencing helps to enrich the transgenic insertion sites and thus is cost-effective and efficient.…”

SLAMF6 deficiency augments tumor killing and skews towards an effector phenotype revealing it as a novel T cell checkpoint

“…pH5, were added to stop the reaction. The DNA purified from the tails was used in PCR reactions for 370 genotyping of mice in the SLAMF6 locus on chromosome 1 (primers adapted from the Jackson laboratories 371 website) and in the Pmel-1 locus on chromosome 2 (Ji et al, 2014). The identification of the genomic 372 insertion site of the Pmel-1 TCR α and β transgenes was performed by next-generation sequencing.…”

Molecular Characterization of Transgenic Events Using Next Generation Sequencing Approach

“…In contrast to conventional PCR and SB methods, NGS has proven to be very sensitive to detect incomplete and multiple integration events [ 7 ]. This technology was also used to characterize transgene insertion sites that were located in complex regions of a genome [ 8 , 9 ]. In plant biotechnology, the number of publications reporting the NGS-based molecular characterization of transgenic events is very limited.…”