FUNCTIONAL GENOMICS OF THE DEVELOPING ENDOCRINE PANCREAS

糖尿病是一个严重的健康问题，在美国影响大约1600万人。 胰腺β细胞缺乏足够的胰岛素产生是I型糖尿病的标志，而在II型糖尿病中，还存在外周胰岛素抵抗。 体内和体外β细胞再生的未来治疗方法将大大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，将提供有关基因表达的复杂模式，管理这一过程的详细信息。本提案的具体目标1是开发标准化和消减的cDNA文库，其富含在发育中的内分泌胰腺中表达的罕见转录物，并确定先前未知转录物的全长序列。 目的2是产生代表数千个胰腺特异性或胰腺富集转录物的cDNA微阵列。 这个微阵列设施将在宾夕法尼亚大学糖尿病中心的范围内建立。 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中获得的标准化新生胰岛cDNA文库，和c）代表罕见胰腺转录物的消减cDNA文库。 后者将利用来自单个细胞的RNA扩增;这是宾夕法尼亚大学首创的，以产生针对单个内分泌细胞类型的特异性cDNA文库。 因此，我们将能够产生代表丰富和罕见的胰腺mRNA的数千个cDNA的复杂微阵列。具体目标3是确定胰腺发育期间和携带影响胰腺发育和功能的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，包括整个胰腺以及单个内分泌细胞谱系。 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，对产生的大量数据进行有效分析。该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新的假设，解决糖尿病的原因和进展。

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.