The goal of this project is to develop techniques for the transgenic expression of altered proteins in retinal neurons. These techniques will be important in establishing the functional role of individual proteins in the retina. With the rapid success of genome sequencing projects, there is increasing information about gene sequences but little information about the functional roles of the proteins encoded by those genes. Transgenic techniques are particularly suitable for the task of deciphering those functional roles. Targeted expression of altered proteins or peptide fragments in particular cell types will allow the physiological action of a protein of interest to be tested in an individual cell. Also, the incorporation of a transgene in which expression of a marker protein is controlled by a target gene's regulatory region can be used to determine the cell types that express a particular gene. To accomplish these goals, methods will be developed that will allow the expression of engineered proteins in retinal neurons. Both conventional bacterial plasmid vectors and bacterial artificial chromosomes (BACs) will be explored for producing DNA constructs that are suitable for transgenesis. The transgenic techniques that are the goal of this project can be combined with electrophysiological and imaging techniques to yield a powerful way of attacking fundamental questions of retinal neurobiology. More broadly, completion of the proposed project will lead to new experimental tools that will be generally useful to retina researchers for the next level of functional analysis, after the completion of genome sequencing initiatives.
