Identification of the genomic insertion site of Pmel-1 TCR α and β Transgenes by next-generation sequencing

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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.

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APA

Ji, Y., Abrams, N., Zhu, W., Salinas, E., Yu, Z., Palmer, D. C., … Restifo, N. P. (2014). Identification of the genomic insertion site of Pmel-1 TCR α and β Transgenes by next-generation sequencing. PLoS ONE, 9(5). https://doi.org/10.1371/journal.pone.0096650

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