Novel Gene Expression Assay in Living Neuronal Subtypes

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DESCRIPTION (provided by applicant): In many diseases the function of subsets of cells is disrupted, e.g. the selective death of dopamine-containing neurons in Parkinson's disease, or the subsets of cells that become cancerous amidst healthy cells. The tissues of the body are complex heterogeneous mixtures of cell types, each with its own gene expression pattern, and concomitant biological function and vulnerability to disease. Comparing the differential gene expression profiles in cells that play different biological roles or have different vulnerability to disease would elucidate how gene expression generates those differences. The limitations of current technologies prevent the achievement of this important goal. We need gene expression markers to identify subpopulations of cells to target those cells for individual biological study. Other assays require many cells, such as biochemical tests and tests to determine an entire gene expression profile. Therefore, we also need techniques to collect many cells that express a subtype marker. We propose here to combine two established technologies to find gene expression markers of subtypes of cells, and to collect populations of cells based on gene expression. Subtypes of neurons are often identified by their characteristic shapes, therefore the technology to find markers for neuron subtypes will relate the shape of a cell to its expression of the gene marker. This will be accomplished by gene gun delivery of fluorescent dye simultaneously with molecules that fluoresce only in the presence of their target mRNA. The dye will fill the neurons to illuminate their shapes, while the gene expression marker will indicate whether its target gene is expressed in that cell. These markers can then be used to target subtypes of cells for individual study. Furthermore, the mixture of marked and unmarked cells in a tissue can be dissociated and sorted using fluorescence-activated cell sorting into groups. This collection of purified populations of cells of individual subtypes will enable the biochemical and gene expression profile studies necessary to understand how gene expression leads to the unique biological function or dysfunction of each cell subtypes. The development of these novel techniques will revolutionize the study of how gene expression generates biological differences.