GENE EXPRESSION IN SINGLE IDENTIFIED CNS CELLS

新技术使我们能够设想有一天我们将例行公事地定义单个细胞的基因表达谱。这在对中枢神经系统(CNS)的研究中尤其重要，在中枢神经系统的研究中，压倒性的复杂性影响了我们对其发育和功能的理解。对于我们对疾病过程的理解，这一点尤其正确。如果我们能够定义正常状态和病理状态之间的基因表达差异，我们将更接近于了解疾病的过程。实现这一目标的两个关键步骤是从单个细胞中制备具有代表性的核酸探针，并开发一种高通量方法来鉴定此类探针中的序列。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不是已知的，因此很难测试其代表性。类似地，存在可允许确定在此类探针制备中存在已知基因和表达序列标签(EST)的方法。我们建议测试几种从单个、已鉴定的细胞中制备代表性探针的方法，并比较使用这些探针的基因图谱方法。我们的策略是比较几乎相同的单个中枢神经系统细胞的图谱。这将使我们能够对重复性或“噪声”进行评分，以确定哪些方法允许在单个细胞中充分表示基因表达。然后，我们计划使用多种方法分离几种不同类型的中枢神经系统细胞，并对这些细胞进行图谱分析。将进行选定细胞类型的电生理记录，以建立RNA表达和蛋白质之间的相关性，并探索在单个细胞中表达的受体和通道的知识对电生理学家固有的好处。

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.