GENE EXPRESSION IN DIABETES BY SUBTRACTIVE CLONING

描述（改编自申请人的摘要）：这是一个竞争 更新申请，将试图确定差异表达 脂肪和肌肉中对噻唑烷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 购买自Affyoung，并将纳入小鼠和人类在 实验方法在具体目标1中，PI将制备转基因小鼠 在脂肪（aP 2启动子）或肌肉（MCK启动子）中靶向过表达 使用含有以下的构建体：i）野生型PPARgamma; ii）pro 115 gln突变体 PPAR γ。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， 在3 T3细胞中， 成纤维细胞iii）合成的ser 114 ala突变。这些老鼠将完全 表型分析此外，在具体的目的2中，小鼠将用和 没有曲格列酮4周，从肌肉、肝脏和脂肪中提取RNA， 来评估差异基因表达。Affytron M19 k芯片将在 它包括19，000个小鼠基因和EST。特异性基因改变 通过cDNA芯片方法鉴定的基因将使用定量实时荧光定量分析法进行确认。 PCR检测。 在具体的目标3中，将使用Cre产生PPARgamma敲除小鼠。 lox-P方法选择性地减少脂肪和肌肉中的PPARgamma。的 实验将涉及PPARgamma floxed/floxed、floxed/-和野生型小鼠 与其他含有Cre转基因的小鼠交配。敲除小鼠将被 用和不用TZD处理，并通过芯片评估差异表达 技术.这些小鼠将研究TZD是否增强胰岛素敏感性 涉及脂肪与肌肉中的主要PPARgamma效应。 在具体目标4中，将对6例2型DM、6例1型DM和6例对照进行 进行代谢表征并进行脂肪和肌肉活组织检查。微分 将通过cDNA基因阵列技术评估这些组织中的表达 使用包含6，800个基因和35，000个基因的Affyphid 6, 800表达芯片， 含有35,000个EST的芯片对于与对照组相比改变2倍的基因， 6对受试者中至少3对的差异表达将通过以下方式确认： 实时定量PCR。此外，6例S1和SG较低的2型DM fs-IVGTT将与6只具有高S1和高SG的后代进行比较， 将血糖正常的肥胖受试者与6名瘦受试者进行比较， 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.