GENE EXPRESSION IN DIABETES BY SUBTRACTIVE CLONING

通过消减克隆表达糖尿病的基因

• 批准号: 6624872
• 负责人: C RONALD KAHN
• 金额: $ 71.87万
• 依托单位: JOSLIN DIABETES CENTER
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-30 至 2003-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6624872
• 关键词: clinical research; diabetes mellitus genetics; differential display technique; gene expression; genetic regulation; genetically modified animals; human subject; insulin dependent diabetes mellitus; insulin sensitivity /resistance; laboratory mouse; laboratory rat; microarray technology; molecular cloning; noninsulin dependent diabetes mellitus; obesity; striated muscles; subtraction hybridization; thiazoles; transfection

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.

描述（改编自申请人的摘要）：这是一个竞争性的 更新申请将尝试识别差异表达 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将采用芯片上的cDNA基因阵列技术 从 Affymetrix 购买，并将小鼠和人类纳入 实验方法。在具体目标1中，PI将准备转基因小鼠 在脂肪（aP2 启动子）或肌肉（MCK 启动子）中定向超表达 使用含有以下成分的构建体：i)野生型PPARgamma； ii) pro115gln 突变体 PPARγ。检测到丝氨酸磷酸化位点的天然突变 4个肥胖杂合子。该突变损害丝氨酸磷酸化导致 3T3 中甘油三酯的表达增强和积累增加 成纤维细胞。 iii) 合成的 ser114ala 突变。这些老鼠将完全 表型。此外，在具体目标 2 中，小鼠将接受以下治疗： 4周不使用曲格列酮，并从肌肉、肝脏和脂肪中提取RNA 评估差异基因表达。 Affymetrix M19k 芯片将 利用其中包括 19,000 个小鼠基因和 EST。特定基因改变 通过cDNA芯片方法鉴定的将使用实时定量来确认 PCR检测。 在具体目标 3 中，将使用 Cre 生成 PPARgamma 敲除小鼠 lox-P 方法选择性减少脂肪和肌肉中的 PPARgamma。这 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。敲除小鼠将是 使用和不使用 TZD 处理，并通过芯片评估差异表达 技术。这些小鼠将探讨 TZD 是否增强胰岛素敏感性 涉及 PPARgamma 对脂肪与肌肉的主要影响。 在具体目标 4 中，将有 6 个类型 2 DM、6 个类型 1 DM 和 6 个对照 代谢特征并进行脂肪和肌肉活检。微分 这些组织中的表达将通过 cDNA 基因阵列技术进行评估 使用包含 6,800 个基因和 35,000 个基因的 Affymetrix 6800 表达芯片 包含 35,000 个 EST 的芯片。与对照相比，基因改变了 2 倍 6 个受试者对中的至少 3 个，差异表达将通过以下方式确认 实时定量PCR。此外，6 个 2 型 DM 具有低 S1 和 SG fs-IVGTT 将与 6 个具有高 S1 和高 SG 的后代进行比较，并且 6 将血糖正常的肥胖者与 6 名瘦受试者进行比较，以进行差异分析 表达于肌肉和脂肪。

项目成果

Effects of phosphorylation on function of the Rad GTPase.

磷酸化对 Rad GTPase 功能的影响。

• DOI: 10.1042/bj3330609
• 发表时间: 1998
• 期刊: The Biochemical journal
• 作者: Moyers,JS;Zhu,J;Kahn,CR
• 通讯作者: Kahn,CR

Muscle-specific PPARgamma-deficient mice develop increased adiposity and insulin resistance but respond to thiazolidinediones.

• DOI: 10.1172/jci17305
• 发表时间: 2003-08
• 期刊: The Journal of clinical investigation
• 作者: A. Norris;Lihong Chen;S. Fisher;I. Szanto;M. Ristow;A. Jozsi;M. Hirshman;E. Rosen;L. Goodyear;F. Gonzalez;B. Spiegelman;C. Kahn

Trinucleotide repeats at the rad locus. Allele distributions in NIDDM and mapping to a 3-cM region on chromosome 16q.

三核苷酸在 rad 基因座重复。

• DOI: 10.2337/diab.44.2.243
• 发表时间: 1995
• 期刊: Diabetes
• 影响因子: 7.7
• 作者: Doria,A;Caldwell,JS;Ji,L;Reynet,C;Rich,SS;Weremowicz,S;Morton,CC;Warram,JH;Kahn,CR;Krolewski,AS
• 通讯作者: Krolewski,AS

Regulation of growth and tumorigenicity of breast cancer cells by the low molecular weight GTPase Rad and nm23.

• 发表时间: 2001-03
• 期刊: Cancer research
• 影响因子: 11.2
• 作者: Yu-Hua Tseng;David Vicent;Jianhua Zhu;Y. Niu;A. Adeyinka;J. Moyers;Peter H Watson;C. Kahn;Y-H T

Insulin receptor substrate 1 binds two novel splice variants of the regulatory subunit of phosphatidylinositol 3-kinase in muscle and brain.

胰岛素受体底物 1 结合肌肉和大脑中磷脂酰肌醇 3-激酶调节亚基的两个新剪接变体。

• DOI: 10.1128/mcb.16.5.2195
• 发表时间: 1996
• 期刊: Molecular and cellular biology
• 影响因子: 5.3
• 作者: Antonetti,DA;Algenstaedt,P;Kahn,CR
• 通讯作者: Kahn,CR