Quantitative PCR for detection of the OT-1 transgene

Kate O Wright,Debbie A Murray,Nicholas I Crispe,Robert H Pierce

doi:10.1186/1471-2172-6-20

Kate O Wright, Debbie A Murray + Show 2 more

Open Access

https://doi.org/10.1186/1471-2172-6-20

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Abstract

BackgroundTransgenic TCR mice are often used experimentally as a source of T cells of a defined specificity. One of the most widely used transgenic TCR models is the OT-1 transgenic mouse in which the CD8+ T cells express a TCR specific for the SIINFEKL peptide of ovalbumin presented on kb. Although OT-1 CD8+ can be used in a variety of different experimental settings, we principally employ adoptive transfer and peptide-driven expansion of OT-1 cells in order to explore the distribution and fate of these antigen-specific OT-1 T cells. We set out to develop a quantitative PCR assay for OT-1 cells in order to assess the distribution of OT-1 CD8+ T cells in tissues that are either intrinsically difficult to dissociate for flow cytometric analysis or rendered incompatible with flow cytometric analysis through freezing or fixation.ResultsWe show excellent correlation between flow cytometric assessment of OT-1 cells and OT-1 signal by qPCR assays in cell dilutions as well as in in vivo adoptive transfer experiments. We also demonstrate that qPCR can be performed from archival formalin-fixed paraffin-embedded tissue sections. In addition, the non-quantitative PCR using the OT-1-specific primers without the real-time probe is a valuable tool for OT-1 genotyping, obviating the need for peripheral blood collection and subsequent flow cytometric analysis.ConclusionAn OT-1 specific qPCR assay has been developed to quantify adoptively transferred OT-1 cells. OT-1 qPCR to determine cell signal is a valuable adjunct to the standard flow cytometric analysis of OT-1 cell number, particularly in experimental settings where tissue disaggregation is not desirable or in tissues which are not readily disassociated

Highlights

Transgenic T cell receptor (TCR) mice are often used experimentally as a source of T cells of a defined specificity
OT-1 Genotyping PCR is a specific and reliable method to determine positive OT-1 mice from wild-type littermates A PCR strategy was developed to identify OT-1 positive transgenic mice, which express a T cell receptor (TCR) specific for the OVA peptide SIINFEKL, from wild-type littermates
Show a marked increase in OT-1 transgene copy number in the OVA treated animals compared to PBS controls (Figure 9). These results further demonstrate the utility of this assay for tissue samples that are difficult to disassociate for flow cytometry and that a migratory OT-1 CD8+ T cell population is present in lung tissue of animals after adoptive transfer experiments

Summary

Introduction

Transgenic TCR mice are often used experimentally as a source of T cells of a defined specificity. Adoptive transfer of CD8+ T cells from MHC Class Irestricted OVA specific T cell receptor (TCR) transgenic mice (OT-1) into host C57BL/6 mice allows for the activated CD8+ T cell population to be tracked and quantified after stimulation with the OVA peptide SIINFEKL [1] using standard flow cytometric analysis. Using this methodology, it has been demonstrated that activated CD8+ T cells undergo migration to the liver where they are trapped and undergo apoptosis [2]. Plasmid pcRTOPO2.1 containing the cloned OT-1 product was used as a positive control (lane 7)

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