Molecular Genetics of a Lens Intrinsic Membrane Protein

晶状体固有膜蛋白的分子遗传学

• 批准号: 6680812
• 负责人: ROBERT L CHURCH
• 金额: $ 38.25万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6680812
• 关键词: DNA footprinting; cataract; cell differentiation; electron microscopy; fiber cell; gel mobility shift assay; gene expression; gene mutation; gene targeting; genetic regulatory element; genetically modified animals; laboratory mouse; lens proteins; membrane proteins; methylation; molecular genetics; oligonucleotides; polymerase chain reaction; transcription factor

DESCRIPTION (provided by applicant): The ocular lens is an avascular, semisolid organ whose function is to refract incoming light upon the retina. Concomitant with this function is a necessity for the lens to remain transparent. A loss of transparency of the lens is usually permanent and is termed a cataract. Cell and organ functions, such as obtaining nutrients necessary for maintaining the healthy, transparent lens state, for handling of cell waste removal, and for maintenance of overall membrane stability are carried out through select and specific proteins or groupings of proteins within the lens cell membrane. Proper controls on expression of these proteins and their subsequent function is essential for lens survival, and maintenance of transparency. The mechanisms regulating these membrane proteins in the lens are presently unknown. The long term goals of this project are to understand the function of the lens fiber cell intrinsic membrane protein MP19 and the regulation of the gene coding for this protein (LIM2). To achieve this goal, we will investigate the following topics: a). Using knockout mouse technology, to ablate the Lim2 gene in order to study the function of MP19. b). Using mammalian expression vectors, the investigate the synthesis of MP19 and various mutations of this coding sequence. Truncations of the MP19 expressed protein will be made to investigate the membrane topology of MP19. Finally, we will use these expression vectors to investigate the putative signal residing in the MP19 molecule which effects transport of MP19 to the cell membrane. c). Investigate functional consequences of point mutations in the Lim2 gene using site-specific RNA/DNA chimeric oligonucleotides (CO). Specific CO will be injected into mouse pronuclei and subsequently implanted in foster mothers. Pups containing successful mutations will be investigated to determine the effect of the mutation on lens development and integrity. These studies will give a better understanding of the role of MP19 in the maintenance of lens transparency and its possible role in cataract formation.

描述（由申请人提供）：眼透镜是一种无血管的半固体器官，其功能是将入射光反射到视网膜上。伴随着该功能，透镜必须保持透明。透镜透明度的丧失通常是永久性的，称为白内障。细胞和器官功能，例如获得维持健康透明的透镜状态所必需的营养物，用于处理细胞废物去除，以及用于维持整体膜稳定性，通过选择和特定的蛋白质或透镜细胞膜内的蛋白质组来进行。适当控制这些蛋白质的表达及其随后的功能对于透镜的存活和透明度的维持是必不可少的。调节透镜中这些膜蛋白的机制目前尚不清楚。本项目的长期目标是了解透镜纤维细胞内膜蛋白MP 19的功能和编码该蛋白的基因（LIM 2）的调控。为了实现这一目标，我们将研究以下主题：a）。利用基因敲除小鼠技术，切除Lim 2基因，研究MP 19的功能。B）。使用哺乳动物表达载体，研究MP 19的合成和该编码序列的各种突变。将对MP 19表达的蛋白进行截短以研究MP 19的膜拓扑结构。最后，我们将使用这些表达载体来研究MP 19分子中存在的影响MP 19转运到细胞膜的假定信号。（c）。使用位点特异性RNA/DNA嵌合寡核苷酸（CO）研究Lim 2基因点突变的功能后果。将特定的CO注射到小鼠原核中，随后植入寄养母亲体内。将研究含有成功突变的幼仔，以确定突变对透镜发育和完整性的影响。 这些研究将使我们更好地了解MP 19在维持透镜透明度中的作用及其在白内障形成中的可能作用。