The development and maintenance of the retina is affected by specific associations between different types of cells. The long range objective of this project is to establish molecular mechanisms by which genetic programming and communication between cells interact to determine the phenotype of Miller glial cells. The proposed research focuses on identifying genetic mechanisms regulating expression of the glutamine synthetase gene. These studies involve gene transfer of intact retinal organ cultures in conjunction with biochemical and cell biological analyses. Previous research established that a 42 nucleotide upstream enhancer element mediates the glucocorticoid induction of the glutamine synthetase gene in transfected retina. This element contains a single binding size for the glucocorticoid receptor as well as an essential binding site for a member of the jun//ATF/CREB family of transcription factors. The first specific aim is to determine whether collaboration between proteins binding at these sites mediates the glucocorticoid response and whether alterations in the functioning of proteins acting at these sites is responsible for altered inducibility during retinal development and in response to disruption of neuronal-glial cell contacts. The second specific aim is to identify cis-acting elements of the glutamine synthetase gene encoding genetic information that positions glutamine synthetase uniquely in Miller glial cells as the differentiated retina emerges from a simple neuroepithelium. The third specific aim is to identify the genetic elements that mediate the over 100-fold rise in GS mRNA that occurs during retinal development and to determine whether interactions between the pigment epithelial cells and the neural retina affect this constitutive activation in vitro. Studies comprising specific aims 1,2 and 3 include gene transfer experiments and are tractable because intact retina can be transfected using electroporation. The fourth specific aim involves a general characterization of the electroporation process and the development of additional methods of retinal gene transfer in vitro. These studies will aid the analysis of the glutamine synthetase gene as well as provide methods to expand the variety of problems in retinal biology that can be addressed using gene transfer and transient expression. The goals of the studies described in this proposal are to achieve a more general appreciation of the underlying mechanisms regulating expression of the glutamine synthetase gene in the retina and to establish general methods that facilitate additional studies of the molecular genetics of retinal gene expression. Targeting the expression of proteins to specific cells is an important component of strategies of gene therapy. This requires an understanding of basic mechanisms and genetic elements regulating gene expression. By characterizing the regulatory elements of a retinal gene and developing additional methods of gene transfer, the research described in this proposal is intended to help make targeted gene therapy a more tenable approach to the treatment of retinal disease.
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