Cell-specific Transcript Profiling in Complex Tissues

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DESCRIPTION (provided by applicant): Remarkable advances have been made over the last decade in our ability to analyze patterns of mRNA expression at the genome-wide level. However, these experimental approaches are limited to pure cell populations. An important challenge that remains is the exploration of the intricate developmental physiological and pathological relationships in expression patterns between multiple cell types in a complex tissue. Often, physical isolation of populations of individual cell types, without altering expression patterns in the process, can be difficult because of intimate anatomical relationships underlying functional heterogeneity in complex organs such as the brain. The proposed experimental approach addresses these challenges in a manner that does not rely on prior cell separations. Furthermore, this approach will assess the levels of transcripts that are being actively translated, which is a more meaningful measurement for defining the physiological attributes of a cell than total transcript levels. This determination will be accomplished by expressing epitope-tagged ribosomal proteins in place of the wild type proteins in cell types of interest. The specifically tagged ribosomes will be isolated from whole tissue homogenates prepared such that the ribosomes continue to carry their associated complement of transcripts from the cell type of origin. This approach will allow analysis of actively translated, cell-specific transcripts from snap-frozen tissue samples, obviating perturbations in transcript patterns arising from prior physical separations of the cells of interest. This methodology should be applicable to any cell type for which cell-specific promoters are available, including specific cells of the immune system and the brain. The purpose of the proposed research is to test the efficacy of this concept, which has broad applicability in research areas such as host responses to infectious disease, cancer biology and neurobiology.