Single cell PCR microarray study of alternative splicing

描述(由申请人提供)：我们感兴趣的是复杂神经系统中的单个神经元是如何相互区分的这个一般性问题。许多细胞表面分子已经在不同的物种中被描述，这些分子具有一个有趣的特性，即有许多可能的形式由单个基因组成，这表明来自单个基因的这种多样性可能是允许不同神经元彼此不同的一般机制。我们关注的是果蝇DSCAM基因，它编码一种对轴突引导至关重要的细胞黏附分子。值得注意的是，38,016个可能的选择性剪接变体允许DSCAM蛋白的免疫球蛋白样域具有非凡的多样性。我们已经开始解决并提议在这次赠款申请中详细解决的一个关键问题是，不同类型的神经元，甚至是特定类型的不同个体神经元，如何利用这种非凡的多样性潜力。我们开发了一种灵敏的单细胞RT-PCR方法，它利用了我们创建的定制微阵列。我们的初步研究使我们估计，每个神经元表达50个或更多不同的mRNA，这些mRNA是从数千个剪接变体中挑选出来的，这些剪接变体具有不同的细胞类型。这可以允许每个小区的DSCAM指令集与其邻居的不同。这些初步结果令人兴奋，因为它们表明我们可以分析小细胞群体和单个神经元，也因为它们已经让我们深入了解DSCAM多样性如何区分给定类型的单个神经元。根据这些结果，我们将： -批判性地评估和扩展我们对果蝇神经元的初步分析 -分析特定种群果蝇神经元中的DSCAM剪接，包括单细胞分析 -使用RNAi在体外研究剪接因子的作用，并研究一个有趣的学习突变体 -通过研究Neurexins，探索哺乳动物神经元在单细胞水平上的替代剪接。

DESCRIPTION (provided by applicant): We are interested in the general question of how individual neurons in a complex nervous system are differentiated from one another. A number of cell surface molecules have been described in various species which share the interesting property of having many possible forms made from a single gene, indicating that such diversity from a single gene could be a general mechanism allowing different neurons to be different from one another. We are focusing on the Drosophila DSCAM gene, which encodes a cell adhesion molecule that is essential for axon guidance. Remarkably, 38,016 possible alternative splice variants allow extraordinary diversity in the immunoglobulin-like domains of the DSCAM protein. A key question that we have begun to address and propose to address in detail in this grant application is how this remarkable potential for diversity is used by different neuron types, and indeed by different individual neurons of a given type. We have developed a sensitive single cell RT-PCR approach, which takes advantage of a custom made microarray that we created. Our preliminary studies lead us to estimate that each neuron expresses 50 or more distinct mRNAs chosen from a spectrum of thousands of splice variants distinctive of its cell type. This can allow every cell's DSCAM repertoire to be different from those of its neighbors. These preliminary results are exciting because they show that we can analyze small cell populations and individual neurons, and because they already have given us insights into the way that DSCAM diversity differentiates single neurons of a given type. Following on these results, we will: -Critically evaluate and extend our preliminary analyses of Drosophila neurons -analyze DSCAM splicing in specific populations of Drosophila neurons including single cell analyses -study the role of splicing factors in vitro using RNAi and study an interesting learning mutant -explore alternative splicing at the single cell level in mammalian neurons by studying neurexins.