Expression data of Sirt1-deficient and sufficient medullary thymic epithelial cells (mTECs)

Medullary thymic epithelial cells play essential role for induction of central self-tolerance. This functional capacity is mediated through a phenomenon known as promiscuous gene expression (pGE) of various tissue-restricted antigen (TRA) genes. pGE was previously shown to be mediated by a single factor called the Autoimmune regulator (Aire), which is specically expressed by mTECs. The aim of this study was to analyze the impact of deacetylase Sirtuin1, which is also highly expressed by mTECs, on mTEC gene expression profile and compare it with the impact of Aire. We used Affymetrix mouse 1ST arrays to analyze the impact of the Sirt1 gene on the gene expression profile of MHC-II high medullary thymic epithelial cells Mature MHC-II high mTECs were flow-sorted from thymi isolated from B6.Sirt1fl/fl, B6.Sirt1fl/flFoxn1-Cre and B6.Aire-/- 6weeks old mice. Typically, 3 mice were pooled to obtain 30-60 thousand sorted mTECs, which were then analyzed by gene expression profiling. Specifically, total RNA was extracted from ~30,000 pooled sorted cells using Trizol. Purified total RNA was then amplified using the MessageAmp RNA kit (Ambion). Biotinylated cRNA was then hybridized to Affymetrix Mouse Gene 1-ST arrays by the genomics core at the Weizmann Institute髓质胸腺上皮细胞（medullary thymic epithelial cells, mTECs）在诱导中枢自身耐受过程中发挥关键作用。这一功能特性通过多种组织限制性抗原（tissue-restricted antigen, TRA）基因的弥散性基因表达（promiscuous gene expression, pGE）现象实现。既往研究表明，弥散性基因表达由单一因子——自身免疫调节因子（Autoimmune regulator, Aire）介导，而该因子仅由mTECs特异性表达。本研究旨在分析同样在mTECs中高表达的去乙酰化酶Sirtuin1对MHC-II高表达髓质胸腺上皮细胞基因表达谱的影响，并将其与Aire的调控作用进行对比。我们采用Affymetrix小鼠1ST基因芯片分析Sirt1基因对MHC-II高表达成熟髓质胸腺上皮细胞的基因表达谱的影响。实验样本取自6周龄的B6.Sirt1fl/fl、B6.Sirt1fl/flFoxn1-Cre及B6.Aire-/-小鼠的胸腺，通过流式细胞术分选出MHC-II高表达的成熟髓质胸腺上皮细胞。通常将3只小鼠的细胞混合以获取30000至60000个分选后的mTECs，随后开展基因表达谱分析。具体实验步骤如下：使用Trizol试剂从约30000个混合分选细胞中提取总RNA，随后采用MessageAmp RNA试剂盒（Ambion公司）对纯化后的总RNA进行扩增，再将生物素标记的cRNA与Affymetrix小鼠基因1-ST芯片进行杂交，该杂交实验由魏茨曼研究所的基因组核心实验室完成。