Signal joint T-cell receptor excision circle assay in miniature swine.
Signal joint T-cell receptor excision circle assay in miniature swine.
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DOI：
10.1097/tp.0b013e31822b9287
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发表时间：
2011-09-27
期刊：
影响因子：
6.2
通讯作者：
Sachs DH
中科院分区：
文献类型：
--
作者：
Vallabhajosyula P;Tena A;Yamada K;Sachs DH
T细胞受体切除环(TREC)的分析已经在人类、灵长类和小鼠模型中被用作胸腺活动的测量，但还没有在偶蹄目动物中描述类似的分析。我们描述了猪信号关节(Sj)TREC分析的发展，并提供了以前在偶蹄目动物中识别它的困难的可能原因。利用人和小鼠sjTREC已知基因组序列的同源性，设计了猪sjTREC的聚合酶链式反应(PCR)引物。来自sjTREC的ψJα侧的引物来自已知的猪序列。利用来自ψJα区的正向引物和来自可能的δ-REC区的反向引物，在猪和羊的两个偶蹄目动物中鉴定了sjTREC。与灵长类TREC的检测不同，最初使用接近δ-REC的类似引物未能产生sjTREC产物。从人和鼠之间的同源区衍生的引物将大约800bpair行进到δ-rec中，从而检测到sjTREC。比较这两个物种之间的sjTREC发现，这两个偶蹄目之间的同源性最高。建立了猪sjTREC的定量聚合酶链式反应(QPCR)分析方法。对两个偶蹄目动物中sjTREC序列的鉴定和分析表明，以前使用已知的sjTREC序列克隆猪TREC的尝试失败了。猪信号联合TREC试验的发展应该能够更直接地量化移植生物学的猪模型中的胸腺活动。
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.