GENE EXPRESSION IN DIABETES BY SUBTRACTIVE CLONING

描述(改编自申请者的摘要)：这是一项竞争 续订申请将尝试识别差异表达 脂肪和肌肉中对噻唑二酮调节敏感的基因 (TZD)和糖尿病。PI将采用带有芯片的cDNA基因阵列技术 从Affymetrix购买，将把老鼠和人类都纳入 实验方法。在特定目标1中，PI将制备转基因小鼠 在脂肪(aP2启动子)或肌肉(MCK启动子)中靶向高表达 使用包含：i)野生型PPARGamma；ii)pro115gln突变体的构建体 PPARGamma。检测到丝氨酸磷酸化位点的这种天然突变。 4例肥胖杂合子。该突变损害丝氨酸磷酸化先导 以增强3T3中甘油三酯的表达和增加积累 成纤维细胞。Iii)人工合成的ser114ala突变。这些老鼠将会完全 表型。此外，在特定目标2中，将对小鼠进行和 停用曲格列酮4周，并从肌肉、肝脏和脂肪中提取RNA 以评估差异基因的表达。Affymetrix M19k芯片将是 其中包括19,000个小鼠基因和EST。特定的基因改变 通过基因芯片方法鉴定将使用实时定量方法进行确认 聚合酶链式反应检测。 在特定的目标3中，将使用Cre产生PPARGamma基因敲除小鼠 LOX-P方法选择性降低脂肪和肌肉中的PPARγ。这个 实验将涉及PPARGamma漂白/漂白、漂白/-和野生型小鼠 与其他携带Cre转基因的小鼠一起饲养。基因敲除的老鼠将会是 TZD治疗前后差异表达的芯片检测 技术这些小鼠将研究TZD是否会增强胰岛素敏感性 在脂肪和肌肉中涉及主要的PPAR伽马效应。 在特定的目标中，将有4名，6名2型糖尿病，6名1型糖尿病和6名对照 具有代谢特征，并接受脂肪和肌肉活组织检查。差动 这些组织的表达将通过cdna基因阵列技术进行评估。 使用包含6,800个基因的Affymetrix 6800表达芯片和35,000个 含有35,000个EST的芯片。与对照组相比，基因改变了2倍 至少6对受试者中的3对，差异表达将通过以下方式确认 实时荧光定量PCR法。此外，6个类型2 DM具有低的S1和SG ON 将FS-IVGTT与6个高S1和高SG的后代和6个 血糖正常的肥胖者将与6名瘦的受试者进行比较，以进行区分 在肌肉和脂肪中的表达。

DESCRIPTION (adapted from the applicant's abstract): This is a competing renewal application that will attempt to identify differentially expressed genes in fat and muscle that are sensitive to regulation by thiazolidenedione (TZD) and in diabetes. The PI will employ cDNA gene array technology with chips purchased from Affymetrix, and will incorporate both mice and humans in the experimental approach. In specific aim 1, the PI will prepare transgene mice with targeted hyperexpression in fat (aP2 promoter) or muscle (MCK promoter) using constructs containing: i) wild type PPARgamma; ii) pro115gln mutant PPARgamma. This native mutation in a serine phosphorylation site was detected in 4 obese heterozygotes. The mutation impairs serine phosphorylation leading to enhanced expressed and an increase in triglyceride accumulation in 3T3 fibroblasts. iii) a synthetic ser114ala mutation. These mice will be fully phenotyped. Furthermore, in specific aim 2, the mice will be treated with and without troglitazone for 4 weeks, and RNA extracted from muscle, liver, and fat to assess differential gene expression. The Affymetrix M19k chip will be utilized which includes 19,000 mouse genes and ESTs. Specific gene alterations identified by cDNA chip approach will be confirmed using quantitative real-time PCR detection. In specific aim 3, PPARgamma knockout mice will be generated using the Cre lox-P approach to selectively reduce PPARgamma in fat and muscle. The experiments will involve PPARgamma floxed/floxed, floxed/-, and wild type mice bred with other mice containing Cre transgenes. The knockout mice will be treated with and without TZD, and differential expression assessed by chip technology. These mice will address whether TZD enhanced insulin sensitivity involves primary PPARgamma effects in fat versus muscle. In specific aim 4, 6 type 2 DM, 6 type 1 DM, and 6 controls will be metabolically characterized and undergo fat and muscle biopsies. Differential expression in these tissues will be assessed by cDNA gene array technology using the Affymetrix 6800 expression chip containing 6,800 genes and the 35,000 chip containing 35,000 ESTs. For genes altered 2-fold compared with controls in at least 3 of the 6 subject pairs, differential expression will be confirmed by real-time quantitative PCR. In addition, 6 type 2 DM having low S1 and SG on fs-IVGTT will be compared with 6 offspring with high S1 and high SG, and 6 normoglycemic obese will be compared with 6 lean subjects, for differential expression in muscle and fat.