REGULATION OF GENE EXPRESSION IN THE DEVELOPING RETINA

视网膜的发育和维持受到特定因素的影响 不同类型细胞之间的联系。的长期目标 这个项目是为了建立遗传基因的分子机制。 细胞之间的编程和通信相互作用，以确定 Miller神经胶质细胞的表型。拟议的研究重点是 确定谷氨酰胺表达的遗传调控机制 合成酶基因。这些研究涉及完整视网膜的基因转移。 结合生化和细胞生物学的器官培养 分析。先前的研究证实，上游的42个核苷酸 增强子元件介导糖皮质激素对谷氨酰胺的诱导 转基因视网膜中的合成酶基因。此元素包含单个 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 各种因素。第一个具体目标是确定协作是否 在这些位点结合的蛋白质之间介导糖皮质激素 以及作用于蛋白的功能是否发生改变 这些部位是视网膜期间诱导性改变的原因。 发展和对神经元-神经胶质细胞接触中断的反应。 第二个具体目标是识别谷氨酰胺的顺式作用元件。 编码定位谷氨酰胺的遗传信息的合成酶基因 作为分化视网膜的Miller神经胶质细胞中唯一的合成酶 从一个简单的神经上皮细胞分化而来。第三个具体目标是 确定调节GS增加100倍以上的遗传因素 在视网膜发育过程中发生的mRNA，并确定 色素上皮细胞与神经视网膜的相互作用 在体外影响这种结构性激活。包含特定内容的研究 目标1、2和3包括基因转移实验，并且易于处理，因为 完整的视网膜可以通过电穿孔技术进行转基因。第四次 具体目标包括电穿孔的一般特征 视网膜基因转移新方法的研究进展 在试管中。这些研究将有助于谷氨酰胺合成酶的分析。 基因，并提供了扩展问题种类的方法 视网膜生物学可以通过基因转移和暂时性研究 表情。本提案中所述研究的目标是 对基本的监管机制有更全面的了解 谷氨酰胺合成酶基因在视网膜中的表达 有助于进一步研究分子的一般方法 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。 这需要了解基本的机制和遗传因素。 调控基因表达。通过描述监管要素的特征 视网膜基因和开发更多的基因转移方法， 这项提案中描述的研究旨在帮助制作靶向基因 治疗是治疗视网膜疾病的一种更可靠的方法.

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.