GENE EXPRESSION IN SINGLE, IDENTIFIED CNS CELLS

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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.