MOLECULAR DISSECTION OF THE ORGAN OF CORTI

柯蒂氏器官的分子解剖

• 批准号: 6471381
• 负责人: Kirk W. Beisel
• 金额: $ 19.39万
• 依托单位: FATHER FLANAGAN'S BOYS' HOME
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-17 至 2003-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6471381
• 关键词: complementary DNA; ear hair cell; functional /structural genomics; gene expression; genetic library; genetically modified animals; laboratory mouse; laser capture microdissection; membrane channels; microarray technology; microprocessor /microchip; molecular cloning; organ of Corti; polymerase chain reaction; technology /technique development

DESCRIPTION (provided by applicant): The mouse inner ear offers an excellent paradigm to characterize and analyze the functional genomics of unique and rare cell types. We propose to utilize the specialized cells of the organ of Corti (OC) for construction and analysis of full-length, microquantity cDNA (mq-cDNA) libraries and the development of an OC UNIGENE set for expression analyses. We wifi utilize our current mq-cDNA protocol with a number of cell procurement strategies to construct representative immortalized full-length cDNA libraries from the cells of the OC with a focus on the supporting cells. In specific aim 1, cochlear hair cells (HCs) provide relatively "known" cell types for assurance of the quality of both the resulting "immortalized" mq-cDNA templates and subsequent mqcDNA libraries. For expression profiling and cell procurement we will test the value of utilizing a Chnra9-GFP transgenic mouse line that express green fluorescent protein in cochlear hair cells. In specffic aim 2, subtracted normalized cell-specific cDNA libraries will be constructed which represent the supporting cells within the OC. These are inner phalangeal, border cells of the inner sulcus, inner pifiar, outer pifiar, Deiters?, and Hensen?s cells, as well as the bordering Claudius? cells. Cell procurement protocols, e.g., microdissection, cell plucking, and if needed laser-capture microscopy, will be used with the mq-cDNA protocol for full-length cDNA library production. GFP-expressing transgenic mice will be used to assist in isolation of specffic types of OC supporting cell. Quality assessment of these cDNAs will be accomplished by using in silico microarray analyses to detect expression of ion channel genes, rare to common housekeeping genes, developmentally expressed genes, cell-specific genes of the OC, and genes expressed in only non-sensory/non-neuronal cells. In specific aim 3 the cell-specific clones derived from aims 1 & 2 will be used to generate a microarray chip containing an OC "UNIGENE" set. Further identification of novel and/or new transcripts wifi be done by the combined use of suppressive subtractive hybridization with microarray analyses. Specific aim 4 will use differential expression analysis to verify the cellular specificity of these clones using the OC chip for in silico microarray analyses in combination with null and spontaneous mutant mice. Extrapolation of the expression profiles may provide insights into the functional characteristics and status of each of these cell types in normal and pathogenic states of the OC.

描述（由申请人提供）：小鼠内耳提供了一个很好的 模式来表征和分析独特和罕见的功能基因组学 细胞类型。我们建议利用科蒂器官的特化细胞 (OC)用于构建和分析全长微量cDNA（mq-cDNA） 文库和开发用于表达分析的OC UNIGENE集。我们 wifi利用我们当前的mq-cDNA协议进行大量细胞采购 构建代表性永生化全长cDNA文库的策略 从OC的细胞中分离出来，重点放在支持细胞上。具体目标 1，耳蜗毛细胞（HC）提供相对“已知”的细胞类型， 确保所得“永生化”mq-cDNA模板的质量 和随后的mqcDNA文库。用于表达谱分析和细胞获取 我们将测试利用Chnra 9-GFP转基因小鼠系的价值， 耳蜗毛细胞表达绿色荧光蛋白。在具体目标2中， 将构建消减的标准化细胞特异性cDNA文库， 代表OC内部的支持细胞。这些是内指骨 内沟边缘细胞、内髓缘细胞、外髓缘细胞、Deiters？、和 汉森？的细胞，以及接壤的克劳迪亚斯？细胞电池采购 协议，例如，显微切割，细胞采摘，如果需要，激光捕获 显微镜，将与全长cDNA文库的mq-cDNA方案一起使用 生产表达GFP的转基因小鼠将用于协助分离 特殊类型的OC支持细胞。这些cDNA的质量评估将 通过使用计算机微阵列分析来检测 离子通道基因，罕见到常见的管家基因，发育表达 基因，OC的细胞特异性基因，以及仅在 非感觉/非神经元细胞。在具体目标3中，细胞特异性克隆 将使用来自AIMS 1和2的DNA来产生微阵列芯片， 一套OC“UNIGENE”新的和/或新的转录本的进一步鉴定 可以通过结合使用抑制性消减杂交与 微阵列分析。具体目标4将使用差异表达分析 为了验证这些克隆的细胞特异性， 结合无效突变体和自发突变体的计算机微阵列分析 小鼠外推的表达谱可以提供洞察到 这些细胞类型中的每一种在正常和 OC的致病状态。