GENE EXPRESSION IN SINGLE, IDENTIFIED CNS CELLS

新技术使我们能够设想有一天我们将常规地确定单个细胞的基因表达谱。这在中枢神经系统（CNS）的研究中尤为重要，因为中枢神经系统的复杂性阻碍了我们对其发育和功能的理解。 这对于我们理解疾病过程尤其如此。 如果我们能够确定正常和病理状态下基因表达的差异，我们将更接近于理解疾病的过程。 实现这一目标的两个关键步骤是从单个细胞制备代表性核酸探针和开发用于鉴定此类探针内的序列的高通量方法。 目前存在从单个细胞制备探针的方法，但由于任何单个细胞表达的所有基因都是未知的，因此难以测试其代表性。 类似地，存在可能允许确定已知基因和表达序列标签（EST）在此类探针制备物中的存在的方法。 我们建议测试几种方法的代表性的探针，从个人，确定的细胞，并比较基因分析方法使用这样的探针。 我们的策略是比较几乎相同的单个CNS细胞的特征。 这将使我们能够对再现性或“噪音”进行评分，以确定哪些方法可以充分代表单个细胞中的基因表达。 然后，我们计划使用多种方法分离几种不同类型的CNS细胞并对这些细胞进行分析。从选定的细胞类型的电生理记录将进行，以建立RNA表达和蛋白质之间的相关性，以及探索在一个单一的细胞中表达的受体和通道的库的知识固有的电生理学家的好处。

New technology has allowed us to envisage a time when we will routinely define the gene expression profile of individual cells. This will be particularly important in the study of the central nervous system (CNS), where overwhelming complexity clouds our understanding of its development and function. This is particularly true for our understanding of disease processes. If we could define gene expression differences among the normal and pathological states, we would much closer to an understanding of the disease process. Two crucial steps towards this goal are the preparation of representative nucleic acid probes from individual cells and the development of a high throughput method for identifying the sequences within such probes. Methods for the preparation of probes from individual cells currently exist, but have been difficult to test for representation as all of the genes expressed by any individual cell are not known. Similarly, methods exist that might allow the determination of the presence of known genes and expressed sequence tags (ESTs) in such probe preparations. We propose to test several methods for the preparation of representative probes from individual, identified cells, and compare gene profiling methods using such probes. Our strategy is to compare profiles from individual CNS cells that are nearly identical. This will allow us to score reproducibility, or "noise", to determine which methods allow for an adequate representation of gene expression in individual cells. We then plan to use multiple methods to isolate several different types of CNS cells and profile those cells. Electrophysiological recordings from selected cell types will be carried out in order to establish a correlation between RNA expression and protein, as well as to explore the benefits to an electrophysiologist inherent in a knowledge of the repertoire of receptors and channels expressed in a single cell.