Signal Joint T-Cell Receptor Excision Circle Assay in Miniature Swine

Abstract

Assays for T cell receptor excision circles (TREC) have been utilized in human, primate, and mouse models as a measure of thymic activity, but no comparable assay has been described in artiodactyls. We describe the development of the porcine signal joint (sj) TREC assay, and provide a likely reason for previous difficulties in its identification in artiodactyls. Utilizing the homology between the known genomic sequences in sjTREC in human and mouse, polymerase chain reaction (PCR) primers were derived for the putative porcine sjTREC. Primers from the ψJα side of the sjTREC were derived from the known porcine sequence. The sjTREC in two artiodactyls, swine and sheep, was identified using forward primers from the ψJα region, and reverse primers from the putative δ-rec region. Unlike in the detection of primate TRECs, initially the use of similar primers close to the δ-rec failed to yield the sjTREC product. Marching about 800 basepairs into δ-rec, primers derived from a homology region between human and mouse led to the detection of sjTREC. Comparing sjTREC amongst the species revealed highest homology between the two artiodactyls. A quantitative PCR (QPCR) assay of porcine sjTREC was also developed. Identification and analysis of the sjTREC sequences in two artiodactyls suggested why previous attempts at cloning the pig TREC using known sjTREC sequences were unsuccessful. The development of the porcine signal joint TREC assay should enable a more direct quantification of thymic activity in porcine models of transplant biology.