MOLECULAR DISSECTION OF THE ORGAN OF CORTI

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DESCRIPTION (provided by applicant): The mouse inner ear offers an excellent paradigm to characterize and analyze the functional genomics of unique and rare cell types. We propose to utilize the specialized cells of the organ of Corti (OC) for construction and analysis of full-length, microquantity cDNA (mq-cDNA) libraries and the development of an OC UNIGENE set for expression analyses. We wifi utilize our current mq-cDNA protocol with a number of cell procurement strategies to construct representative immortalized full-length cDNA libraries from the cells of the OC with a focus on the supporting cells. In specific aim 1, cochlear hair cells (HCs) provide relatively "known" cell types for assurance of the quality of both the resulting "immortalized" mq-cDNA templates and subsequent mqcDNA libraries. For expression profiling and cell procurement we will test the value of utilizing a Chnra9-GFP transgenic mouse line that express green fluorescent protein in cochlear hair cells. In specffic aim 2, subtracted normalized cell-specific cDNA libraries will be constructed which represent the supporting cells within the OC. These are inner phalangeal, border cells of the inner sulcus, inner pifiar, outer pifiar, Deiters?, and Hensen?s cells, as well as the bordering Claudius? cells. Cell procurement protocols, e.g., microdissection, cell plucking, and if needed laser-capture microscopy, will be used with the mq-cDNA protocol for full-length cDNA library production. GFP-expressing transgenic mice will be used to assist in isolation of specffic types of OC supporting cell. Quality assessment of these cDNAs will be accomplished by using in silico microarray analyses to detect expression of ion channel genes, rare to common housekeeping genes, developmentally expressed genes, cell-specific genes of the OC, and genes expressed in only non-sensory/non-neuronal cells. In specific aim 3 the cell-specific clones derived from aims 1 & 2 will be used to generate a microarray chip containing an OC "UNIGENE" set. Further identification of novel and/or new transcripts wifi be done by the combined use of suppressive subtractive hybridization with microarray analyses. Specific aim 4 will use differential expression analysis to verify the cellular specificity of these clones using the OC chip for in silico microarray analyses in combination with null and spontaneous mutant mice. Extrapolation of the expression profiles may provide insights into the functional characteristics and status of each of these cell types in normal and pathogenic states of the OC.