Lynch Heather E, Sempowski Gregory D
Duke Human Vaccine Institute, Duke University Medical Center, Durham, NC, USA.
Methods Mol Biol. 2013;979:147-59. doi: 10.1007/978-1-62703-290-2_12.
This chapter provides protocols necessary for quantifying human, mouse, and nonhuman primate signal joint T cell receptor excision circles (sjTRECs) produced during T cell receptor alpha (TCRA) gene rearrangement. These non-replicated episomal circles of DNA are generated by the recombination process used to produce antigen-specific T cell receptors. The number of sjTRECs per mg of thymus tissue or per 100,000 lysed cells has been shown to be a molecular marker of thymopoiesis and naïve T cells. This technology is beneficial to investigators interested in quantitating the level of naïve T cell production occurring under various circumstances in a variety of systems, and complements traditional phenotypic analyses of thymopoiesis. This chapter specifically describes procedures required for rapid detection and quantitation of sjTRECs in thymus tissue or isolated cells using real-time quantitative polymerase chain reaction (PCR). The sjTREC assay system comprises species-specific forward and reverse primers for amplification of a unique site on the T cell receptor δ (TCRD) sjTREC, a fluorescently labeled (FAM/ZEN/IABkFQ) species-specific real-time probe, and a species-specific sjTREC DNA plasmid standard for quantitation.
本章提供了对在T细胞受体α（TCRA）基因重排过程中产生的人、小鼠和非人灵长类信号联合T细胞受体切除环（sjTRECs）进行定量所需的实验方案。这些未复制的环状DNA是通过用于产生抗原特异性T细胞受体的重组过程生成的。每毫克胸腺组织或每100,000个裂解细胞中sjTRECs的数量已被证明是胸腺生成和幼稚T细胞的分子标志物。该技术对有兴趣在各种系统中定量不同情况下幼稚T细胞产生水平的研究人员有益，并补充了胸腺生成的传统表型分析。本章具体描述了使用实时定量聚合酶链反应（PCR）快速检测和定量胸腺组织或分离细胞中sjTRECs所需的程序。sjTREC检测系统包括用于扩增T细胞受体δ（TCRD）sjTTREC上独特位点（unique site）的物种特异性正向和反向引物、荧光标记（FAM/ZEN/IABkFQ）的物种特异性实时探针，以及用于定量的物种特异性sjTREC DNA质粒标准品。
