REGULATION OF GENE EXPRESSION IN THE DEVELOPING RETINA

视网膜的发育和维持受到特定的 不同类型细胞之间的联系。 长期目标： 这个项目是建立遗传学的分子机制， 编程和细胞之间的通信相互作用，以确定 米勒神经胶质细胞的表型。 拟议的研究重点是 鉴定调节谷氨酰胺表达的遗传机制 合成酶基因 这些研究涉及基因转移的完整视网膜 器官培养结合生物化学和细胞生物学 分析。 先前的研究证实， 增强子元件介导糖皮质激素对谷氨酰胺的诱导 合成酶基因转染的视网膜。 此元素包含一个 糖皮质激素受体的结合大小以及必需的 jun//ATF/CREB转录家族成员的结合位点 因素 第一个具体目标是确定合作是否 在这些位点结合的蛋白质之间介导糖皮质激素 反应，以及是否改变蛋白质的功能， 这些位点负责视网膜病变过程中的诱导改变， 发育和响应神经元-神经胶质细胞接触的破坏。 第二个具体目标是鉴定谷氨酰胺的顺式作用元件， 合成酶基因，其编码定位谷氨酰胺的遗传信息 在分化的视网膜等米勒神经胶质细胞中， 由简单的神经上皮细胞形成。 第三个具体目标是 确定介导GS增加100倍以上的遗传元件 在视网膜发育过程中发生的mRNA，并确定是否 色素上皮细胞与神经视网膜的相互作用 在体外影响这种组成性激活。 研究包括特定的 目标1、2和3包括基因转移实验，并且是易于处理的，因为 可以使用电穿孔转染完整视网膜。 第四 具体目的涉及电穿孔的一般表征 视网膜基因转移的过程和其他方法的发展 体外 这些研究将有助于谷氨酰胺合成酶的分析 基因，并提供方法来扩大各种问题， 视网膜生物学，可以解决使用基因转移和瞬时 表情 本提案所述研究的目标是 更全面地了解监管的基本机制 谷氨酰胺合成酶基因在视网膜中的表达， 促进分子的额外研究的一般方法 视网膜基因表达的遗传学 靶向蛋白质的表达 针对特定细胞是基因治疗策略的重要组成部分。 这需要了解基本机制和遗传要素 调节基因表达。 通过描述 视网膜基因和开发其他基因转移方法， 这项提案中描述的研究旨在帮助使靶基因 视网膜疾病的治疗方法有哪些？

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.