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.
Highlights
Transgenic animal models are indispensable resources for studies of gene function and disease
There were no notable differences of intensity or frequency between homozygous and heterozygous cells stained with the antibody specific for the Vb13 chain of pmel-1 T cell receptor (TCR) (Figure 1, upper panel)
This observation allow us to distinguish pmel-1 homozygosity or heterozygosity, the requirements of FACS analysis of naıve CD8+ T cells, the cost of tetramers, and the inability to perform this type of assay reliably on neonatal mice limit the application of this method
Summary
Transgenic animal models are indispensable resources for studies of gene function and disease. Their construction often involves large bacterial or yeast artificial chromosomes, which are used to assemble the transgene [1]. Many transgenic lines remain poorly characterized, and the method for generating these transgenic animals (i.e. the injection of genetic material into the pro-nucleus of a fertilized egg) results in the random integration of foreign transgenic DNA into the genome [1]. Transgenic animals are commonly evaluated by Southern blot to determine gene incorporation [1]. The site of integration, the possibility of rearrangements of the transgene and potential deletions of nonlethal native DNA at the site of integration remain unknown for most transgenic lines. Random and unknown integration of transgenes can affect the behavior of transgenic mice in unpredictable ways [1]
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