FUNCTIONAL GENOMICS OF THE DEVELOPING ENDOCRINE PANCREAS

糖尿病是一个严重的健康问题，影响着美国大约1600万人。胰腺细胞缺乏足够的胰岛素生成是I型糖尿病的标志，而II型糖尿病还存在外周胰岛素抵抗。未来体内和体外β细胞再生的治疗方法将受益于对胰岛发育和分化的全面了解。我们建议建立一个功能基因组学资源，提供有关控制这一过程的复杂基因表达模式的详细信息。本提案的具体目标1是开发标准化和减法的cDNA文库，丰富发育中的内分泌胰腺中表达的罕见转录本，并确定以前未知转录本的全长序列。目的2是生成代表数千个胰腺特异性或胰腺富集转录本的cDNA微阵列。这个微阵列设备将建立在宾夕法尼亚大学糖尿病中心的背景下。这些微阵列的克隆将从三个来源获得：a)用胰腺特异性探针筛选的现有的市售微阵列，b)在Aim 1中获得的规范化新生儿胰岛cDNA文库，以及c)代表罕见胰腺转录物的减去cDNA文库。后者将利用单细胞的RNA扩增；这是宾夕法尼亚大学首创的，用于生成针对个体内分泌细胞类型的cDNA文库。因此，我们将能够生成一个由数千个cdna组成的复杂微阵列，这些cdna代表了丰富的和罕见的胰腺mrna。具体目的3是确定胰腺发育过程中以及携带影响胰腺发育和功能突变的小鼠中数千个基因的表达谱。我们将利用目标2中产生的微阵列来确定内分泌胰腺发育过程中的表达谱，包括整个胰腺和个体内分泌细胞系。我们将利用宾夕法尼亚大学生物信息学中心的计算机资源对生成的大量数据进行有效的分析。这个生物信息学数据库，以及微阵列，将提供给一般研究界。建立这些最先进的技术，并将其应用于内分泌胰腺，将使主要研究人员能够制定和测试解决糖尿病病因和进展的新假设。

Diabetes mellitus is a significant health problem, affecting approximately 16 million people in the United States. Loss of sufficient insulin production by the pancreatic beta cell is the hallmark of type I diabetes, while in type II diabetes there is in addition peripheral insulin resistance. Future therapeutic approaches of regeneration of beta cells both in vivo and in vitro will benefit greatly from a complete understanding of the development and differentiation of the pancreatic islets. We propose to establish a functional genomics resource that will provide detailed information about the complex patterns of gene expression that govern this process. Specific aim 1 of this proposal is to develop normalized and subtracted cDNA libraries enriched for rare transcripts expressed in the developing endocrine pancreas and to determine full length sequences of previously unknown transcripts. Aim 2 is to generate cDNA microarrays representing thousands of pancreas- specific or pancreas-enriched transcripts. This microarray facility will be established within the context of the University of Pennsylvania Diabetes Center. Clones for these microarrays will be obtained from three sources: a) existing, commercially available microarrays screened with pancreas specific probes, b) the normalized newborn islet cDNA library obtained in Aim 1, and c) subtracted cDNA libraries representing rare pancreatic transcripts. The latter will take advantage of RNA amplification from single cells; which was pioneered at Penn, to generate cDNA libraries specific for the individual endocrine cell types. Thus we will be able to generate a complex microarray of thousands of cDNAs which represent both abundant and rare pancreatic mRNAs. Specific aim 3 is to determine the expression profile of thousands of genes during pancreatic development and in mice carrying mutations that affect pancreatic development and function. We will utilize the microarrays generated in aim 2 to determine expression profiles during the development of the endocrine pancreas, both in the entire pancreas as well as in individual endocrine cell lineages. We will utilize the computer resources of the University of Pennsylvania Bioinformatics Center for the efficient analysis of the large body of data generated. This bioinformatics database, as well as the microarrays, will be made available to the general research community. Establishing these state-of-the-art technologies, and applying them to the endocrine pancreas, will enable principal investigators to formulate and test novel hypotheses that address the causes and progression of diabetes.