FUNCTIONAL GENOMICS OF THE DEVELOPING ENDOCRINE PANCREAS

本提案的总体目标是鉴定和表征参与胰腺内分泌发育和功能的基因。这包括与生成胰岛和胰腺有关的基因，以及定义导致1型和2型糖尿病患病状态的生理功能和功能障碍的基因。我们建议利用人类和小鼠的胚胎和成年胰腺组织构建全面的cDNA文库。这些文库将用于制备cDNA芯片（微阵列），作为基因活性的定量和标准化分析。胰腺芯片的可用性将使研究人员能够在多种情况下（发育、癌症和糖尿病）检测基因表达，从而为基因分析和发现提供有效的方法。获得DNA微阵列所需的步骤将由四个实验室完成：哈佛大学的D. Melton；B. Brownstein， A. Permutt和华盛顿大学基因组测序中心（GSC）。项目将分为四个主要部分：1)cDNA文库的构建和寡核苷酸杂交归一化（Melton）； 2)代表性cDNA克隆的测序和公共数据库的保存（Permutt, Genome Sequencing Center， I.M.A.G.E. consortium）；3)。cDNA芯片的制备（Permutt, Brownstein）；4)初步实验，以确定微阵列在胰腺发育和糖尿病研究中的可靠性和实用性（Melton, Permutt, Brownstein）。我们建议建立4个库，包括小鼠和人类不同胚胎阶段的胰腺库，以及成年小鼠和人类的胰腺和胰岛库。每个文库将排列大约50,000个克隆，根据复杂程度，将对多达10,000个克隆进行测序。每个文库的所有50,000个cdna将被标记在DNA微阵列上并进行试点实验。所有cDNA克隆将存入I.M.A.G.E.联盟，所有序列将存入UniGene （NCBI）供公众立即查阅。至少有一家公司（圣路易斯的基因组系统公司）已经表达了将胰岛cdna整合到他们的表达微芯片中的愿望，以供研究界使用。

The overall aim of this proposal is to identify and characterize genes involved in pancreatic endocrine development and function. This includes genes implicated in generating islets and the pancreas as well as genes defining the physiological function and dysfunction leading to the diseased states in type 1 and type 2 diabetes. We propose to construct comprehensive cDNA libraries from both humans and mice, using embryonic and adult pancreatic tissues. These libraries will be used to prepare cDNA chips (microarrays) that will serve as quantitative and standardized assays for gene activity. The availability of pancreatic chips will enable researchers to assay gene expression in numerous contexts (development, cancer, and diabetes) and thereby provide an effective method for gene analysis and discovery. The steps necessary to acquire DNA microarrays will be accomplished by four labs: D. Melton, Harvard; B. Brownstein, A. Permutt, and the Genome Sequencing Center (GSC), Washington University. The project will be divided into four general parts: 1) Construction of cDNA libraries and normalization by oligo hybridization (Melton), 2) Sequencing of representative cDNA clones and depositing in public databases (Permutt, Genome Sequencing Center, I.M.A.G.E. consortium); 3). Preparation of cDNA microarrays (Permutt, Brownstein); 4) Pilot experiments to ascertain the reliability and utility of microarrays for studies of pancreatic development and diabetes (Melton, Permutt, Brownstein) We propose to construct 4 libraries, pancreas pooled from various embryonic stages for mouse and human, and pooled pancreas and islet libraries from adult mouse and human. Approximately 50,000 clones will be arrayed from each library, and depending on complexity, up to 10,000 will be sequenced. All 50,000 cDNAs for each library will be spotted on DNA microarrays and pilot experiments conducted. All cDNA clones will be deposited with the I.M.A.G.E. Consortium, and all sequences placed in UniGene (NCBI) for immediate public access. At least one company (Genome Systems, St. Louis) has expressed the desire to incorporate the islet cDNAs in their expression microchips for the research community.