Regulation of Rhodopsin Expression

视紫红质表达的调节

基本信息

批准号：
7686478
负责人：
Donald J. Zack
金额：
$ 7.5万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1992
资助国家：
美国
起止时间：
1992-08-01 至 2012-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7686478
关键词：
A Mouse Address Alternative Splicing Area Binding Bioinformatics Biological Biological Assay Biological Models Cells Collaborations DNA DNA Binding DNA Sequence Deoxyribonuclease I Development Disease Electroporation Elements Engineering Funding Gene Expression Gene Expression Regulation Genes Grant Histologic Human Hybrids Hypersensitivity In Vitro Knockout Mice Lead Length Libraries Maps Mus Mutant Strains Mice Mutation Nucleic Acid Regulatory Sequences Numbers Opsin Pattern Photoreceptors Physiological Promoter Regions Protein Isoforms RNA Processing RNA Splicing Rattus Regulation Regulator Genes Regulatory Element Reporter Research Design Resolution Response Elements Retina Retinal Retinal Cone Retinal Degeneration Retinal Diseases Retinitis Pigmentosa Retinoblastoma Rhodopsin Screening procedure Small Interfering RNA Technology Transactivation Transfection Transgenic Mice Vertebrate Photoreceptors Work Yeasts base cDNA Expression cell type expression cloning gene therapy insight interest loss of function man mouse model promoter research study success transcription factor

项目摘要

DESCRIPTION (provided by applicant): Retinal development, function, and disease are, to a significant degree, controlled by the pattern of genes expressed by the cells of the retina. In an effort to better understand the mechanisms regulating photoreceptor gene expression, we have been studying rod and cone opsin gene regulation as model systems. Using a variety of approaches, we, and others, have defined some of the DNA elements important for rod- and cone-specific expression, have identified and cloned some of the transcription factors that bind to these DNA elements, and have shown that mutations in some of these factors can both interfere with normal photoreceptor development in the mouse and can cause retinal degeneration in man. This application for continued funding of these studies proposes to continue and broaden this work. The proposed work includes four aims: 1) more fully characterize several already cloned factors that are candidate molecules for regulating the various cone opsins; 2) further develop and utilize in vitro retinal electroporation technology so as to assess the biological activity of rod and cone opsin regulatory elements in a more physiological milieu, and in a complementary fashion use a high-throughput PCR-based DNase I hypersensitivity assay to identify regions of regulatory interest; 3) utilize a high- throughput transient transfection-based assay together with libraries of transcription factor expression clones to identify factors that regulate the response elements identified in "2"; and 4) use a combined bioinformatic and experimental approach to identify genes that are differentially expressed in the retina, and then determine how expression of these retina-specific splice isoforms varies during development, during degeneration in the rd1 mouse, and in three mouse models that express altered forms of three pre-RNA processing factors (PRPF 3, 8 and 31) that when altered in humans cause retinitis pigmentosa. Together, these studies will hopefully provide new insights into the mechanisms regulating photoreceptor gene expression, which in turn will aid in understanding how abnormalities in gene expression can cause retinal disease, and help in the development of more cell-type specific gene therapy approaches.

描述（由申请人提供）：视网膜发育，功能和疾病在很大程度上受视网膜细胞表达的基因模式控制。为了更好地理解调节感光基因表达的机制，我们一直在研究杆和锥蛋白基因调节作为模型系统。使用多种方法，我们和其他方法定义了一些对于杆和锥体特异性表达重要的DNA元素，已经鉴定并克隆了与这些DNA元素结合的一些转录因子，并表明突变中的突变中的突变其中一些因素都可能干扰小鼠的正常感光体发育，并且可能导致人类的视网膜变性。这些研究的持续资金申请建议继续并扩大这项工作。拟议的工作包括四个目的：1）更充分地表征了几个已经克隆的因素，这些因素是调节各种锥体蛋白的候选分子； 2）进一步开发和利用体外视网膜电穿孔技术，以评估更生理的环境中的杆和锥蛋白的调节元件的生物学活性，并以互补的方式使用高通量的PCR PCR I超紧敏测定，以识别监管兴趣地区； 3）利用基于高吞吐量的瞬态转染测定法与转录因子表达克隆的库来识别调节“ 2”中鉴定的响应元件的因素； 4）使用合并的生物信息学和实验方法鉴定在视网膜中差异表达的基因，然后确定这些视网膜特异性剪接同工型在发育过程中，RD1小鼠变性期间以及三种小鼠模型中的表达如何变化。当人类变化时会导致色素性视网膜炎时，三种前RNA加工因子的明确形式（PRPF 3、8和31）。这些研究共同有望提供有关调节光感受器基因表达的机制的新见解，这反过来又有助于了解基因表达中的异常如何引起视网膜疾病，并有助于开发更多细胞型特异性基因治疗方法。