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

胸腺髓质上皮细胞（medullary thymic epithelial cells, mTECs）在中枢自身耐受的诱导过程中发挥至关重要的作用。这一功能特性通过多种组织限制性抗原（tissue-restricted antigen, TRA）基因的异位基因表达（promiscuous gene expression, pGE）现象得以实现。既往研究表明，pGE由单一转录因子自身免疫调节因子（Autoimmune regulator, Aire）介导，且该因子仅在mTECs中特异性表达。本研究旨在探究同样在mTECs中高表达的去乙酰化酶Sirtuin1对mTEC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高表达胸腺髓质上皮细胞的基因表达谱的影响。从6周龄的B6.Sirt1fl/fl、B6.Sirt1fl/flFoxn1-Cre及B6.Aire–/–小鼠中分离胸腺，通过流式细胞术分选出成熟的MHC-II高表达mTECs。通常将3只小鼠的细胞混合以获取3~6万个分选后的mTECs，随后对其进行基因表达谱分析。具体操作如下：使用Trizol试剂从约3万个混合分选的细胞中提取总RNA；纯化后的总RNA通过MessageAmp RNA试剂盒（Ambion公司）进行扩增；最后由魏茨曼研究所基因组核心实验室将生物素标记的cRNA与Affymetrix小鼠基因1-ST芯片进行杂交。