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

Author(s): Ji Y,  Abrams N,  Zhu W,  Salinas E,  Yu Z,  Palmer DC,  Jailwala P,  Franco Z,  Roychoudhuri R,  Stahlberg E,  Gattinoni L,  Restifo NP

Journal: PLoS One

Date: 2014

Major Program(s) or Research Group(s): CBRG

PubMed ID: 24827921

PMC ID: PMC4020793

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.