Quantitative PCR for detection of the OT-1 transgene.
BACKGROUND
Transgenic 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.


RESULTS
We 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.


CONCLUSIONS
An 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.
Wright
Kate O
KO
Department of Pathology, University of Rochester Medical Center, Rochester, NY 14642, USA. KateWright@frontiernet.net
Murray
Debbie A
DA
Crispe
Nicholas I
NI
Pierce
Robert H
RH
D019264:Adoptive Transfer; D000818:Animals; D001483:Base Sequence; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology* / Q000637:transplantation; D004527:Egg Proteins / Q000276:immunology*; D005434:Flow Cytometry; D006183:H-2 Antigens / Q000276:immunology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D010047:Ovalbumin / Q000276:immunology*; D010446:Peptide Fragments; D016133:Polymerase Chain Reaction / Q000379:methods*; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics* / Q000276:immunology; D012680:Sensitivity and Specificity; D037182:T-Cell Antigen Receptor Specificity; D019076:Transgenes; D050441:VDJ Exons
D004527:Egg Proteins; D006183:H-2 Antigens; C032876:H-2Kb protein, mouse; C110366:OVA-8; D010446:Peptide Fragments; D016693:Receptors, Antigen, T-Cell, alpha-beta; D010047:Ovalbumin