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: 6838761
• 负责人: ROBERT L CHURCH
• 金额: $ 34.43万
• 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/6838761
• 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）：晶状体是一种无血管的半实体器官，其功能是将入射光折射到视网膜上。伴随这一功能的是镜片必须保持透明。晶状体失去透明度通常是永久性的，称为白内障。细胞和器官的功能，如获得维持健康透明晶状体状态所需的营养物质，处理细胞废物的清除，以及维持整体膜稳定性，都是通过晶状体细胞膜内选择和特定的蛋白质或蛋白质组来实现的。适当控制这些蛋白的表达及其后续功能对晶状体存活和维持透明度至关重要。晶状体中调节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晶状体纤维细胞内在膜蛋白MP19的功能及其基因编码（LIM2）的调控。为了实现这一目标，我们将调查以下主题：a)。采用敲除小鼠技术，将Lim2基因敲除，以研究MP19的功能。b)。利用哺乳动物表达载体，研究了MP19的合成及其编码序列的各种突变。MP19表达蛋白的截短将用于研究MP19的膜拓扑结构。最后，我们将使用这些表达载体来研究MP19分子中可能存在的影响MP19向细胞膜运输的信号。c)。利用位点特异性RNA/DNA嵌合寡核苷酸（CO）研究Lim2基因点突变的功能后果。将特定的CO注射到小鼠原核中，随后植入养母体内。将研究含有成功突变的幼崽，以确定突变对晶状体发育和完整性的影响。