Gene expression in the pancreatic lymph node of Deaf1 knock out mice vs. BALB/c controls

A microarray study performed in the pancreatic lymph nodes of Deaf1 knock-out and BALB/c control mice to identify genes that are regulated by the transcriptional regulator Deaf1. These experiments constitute a portion of the study described below: Abstract: Type 1diabetes (T1D) can result from a breakdown in peripheral tolerance which is controlled by peripheral tissue antigen (PTA) expression in lymph nodes. Here, we identified a transcriptional regulator, deformed epidermal autoregulatory factor 1 (Deaf1), which regulates the expression of various PTAs in the pancreatic lymph node (PLN). We found, by microarray, that Deaf1 controls the expression of ~600 genes in the PLN. In the non-obese diabetic (NOD) mouse model of T1D, we identified a wild-type form of Deaf1 (DF1) and a truncated alternatively spliced variant (DF1-VAR1) that hetero-dimerizes with and decreases the transcriptional activity of DF1. The expression of DF1 correlates with the expression of various pancreatic PTAs such as insulin, and during the onset of destructive insulitis in NOD mice, DF1 expression is downregulated, while the DF1-VAR1 expression is upregulated in the PLN. A reduction in DF1-controlled PTA expression in the PLN, leading to decreased peripheral tolerance, could underlie the pathogenesis of NOD disease. Deaf1-KO mice (4 wk old) and age-matched BALB/c control mice were sacrificed, and the PLN were removed and immediately homogenized in Trizol Reagent. RNA was extracted in Trizol and then purified using the RNeasy kit (Qiagen). RNA quality was assessed using the Agilent RNA 6000 Nano Reagents, RNA Nano chips, and the Agilent 2100 bioanalyzer (Agilent Technologies), according to manufacturer's instructions. Control and Deaf1-KO mouse RNA was amplified, labeled with Cy3 and Cy5, respectively, and combined with spike A and spike B mix, respectively, using the Agilent low RNA input fluorescence linear amplification kit (Agilent Technologies). The amplified cRNA was purified using the RNeasy kit (Qiagen), and specific activity was determined with the NanoDrop 1000 spectrophotometer (Thermo Scientific). Samples were prepared with the gene expression hybridization kit (Agilent Technologies) and two color microarrays were performed using the whole mouse genome (4x44K) Oligo microarray kit, according to manufacturer's instructions. Microarray chips were washed and scanned using the DNA microarray scanner (Agilent Technologies). Data was processed with Feature Extraction Software (Agilent Technologies), and analyzed using GeneSpring GX 7.3 Software (Agilent Technologies). This submission shows the data obtained from two individual Deaf1 knockout mice measured against a pool of 4 BALB/c control mice.本研究针对Deaf1基因敲除（Deaf1 knock-out）小鼠与BALB/c对照小鼠的胰腺淋巴结开展微阵列（microarray）实验，旨在鉴定受转录调控因子Deaf1调控的基因。本实验为下述研究的一部分： 摘要：1型糖尿病（Type 1 diabetes, T1D）可由外周耐受崩溃引发，而外周耐受受淋巴结内外周组织抗原（peripheral tissue antigen, PTA）的表达调控。本研究中，我们鉴定出一种转录调控因子——变形表皮自调节因子1（deformed epidermal autoregulatory factor 1, Deaf1），其可调控胰腺淋巴结（pancreatic lymph node, PLN）内多种外周组织抗原的表达。通过微阵列实验，我们发现Deaf1可调控胰腺淋巴结内约600个基因的表达。在1型糖尿病的非肥胖糖尿病（non-obese diabetic, NOD）小鼠模型中，我们鉴定出Deaf1的野生型形式（DF1）与一种截短型可变剪接变体（DF1-VAR1），后者可与DF1形成异二聚体并降低DF1的转录活性。DF1的表达与多种胰腺外周组织抗原（如胰岛素）的表达呈正相关；在非肥胖糖尿病小鼠发生破坏性胰岛炎的进程中，胰腺淋巴结内DF1的表达被下调，而DF1-VAR1的表达则被上调。胰腺淋巴结内DF1调控的外周组织抗原表达减少，进而导致外周耐受降低，这可能是非肥胖糖尿病小鼠发病的潜在机制。 我们选取4周龄的Deaf1基因敲除小鼠与同月龄的BALB/c对照小鼠实施安乐死，摘取其胰腺淋巴结并立即置于Trizol试剂（Trizol Reagent）中进行匀浆。采用Trizol法提取总RNA，随后使用RNeasy试剂盒（RNeasy kit, Qiagen）对RNA进行纯化。按照制造商说明书，使用Agilent RNA 6000 Nano试剂（Agilent RNA 6000 Nano Reagents）、RNA Nano芯片（RNA Nano chips）与Agilent 2100生物分析仪（Agilent 2100 bioanalyzer, Agilent Technologies）对RNA质量进行检测。分别使用Cy3与Cy5对对照小鼠与Deaf1基因敲除小鼠的RNA进行标记与扩增，并分别加入spike A与spike B混合液，实验采用Agilent低RNA输入荧光线性扩增试剂盒（Agilent low RNA input fluorescence linear amplification kit, Agilent Technologies）。扩增后的互补RNA（cRNA）使用RNeasy试剂盒（RNeasy kit, Qiagen）进行纯化，并使用NanoDrop 1000分光光度计（NanoDrop 1000 spectrophotometer, Thermo Scientific）测定其比活性。按照制造商说明书，使用基因表达杂交试剂盒（gene expression hybridization kit, Agilent Technologies）制备样本，并采用全小鼠基因组（4x44K）寡核苷酸微阵列试剂盒（whole mouse genome (4x44K) Oligo microarray kit）开展双色微阵列实验。使用DNA微阵列扫描仪（DNA microarray scanner, Agilent Technologies）对芯片进行洗涤与扫描。数据采用Feature Extraction软件（Feature Extraction Software, Agilent Technologies）进行处理，并使用GeneSpring GX 7.3软件（GeneSpring GX 7.3 Software, Agilent Technologies）进行分析。本提交数据集包含2只独立的Deaf1基因敲除小鼠的检测数据，对照数据取自4只BALB/c对照小鼠的混合样本。