We wish to identify regulatory elements involved in brain-specific gene expression using the GABAA receptor alpha 6 subunit gene; this gene is uniquely expressed in differentiated cerebellar granule cells. A comparative approach is to be employed to identify granule cell-specific enhancer elements on the basis that outside the coding region unconstrained sequences have had a maximum time to randomize during the 430 million years seperating teleost-fish and mammals, thus sequences involved in spatially regulated gene expression will be conserved. Exploiting the conservation of cell type organisation in the vertebrate cerebellum, we demonstrated that the alpha 6 expression pattern is conserved in granule cells of birds and fish. Taking advantage of the compact genome of the pufferfish (Fugu rubnpes) we will now inject pufferfish alpha6 constructs into oocytes and use embryonic stem cell technology for a more efficient way of analyzing reporter gene expression in chimeric mice. Combined with the application of comparative genome sequencing, we hope to identify granule cell-specific regulatory elements.