MOLECULAR DISSECTION OF THE ORGAN OF CORTI

柯蒂氏器官的分子解剖

• 批准号: 7093403
• 负责人: Kirk W. Beisel
• 金额: $ 8.04万
• 依托单位: CREIGHTON UNIVERSITY
• 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 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7093403
• 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(由申请者提供)：老鼠内耳提供了一个极好的 描述和分析独特和稀有动物功能基因组学的范式 单元类型。我们建议利用科尔蒂器官的特化细胞 (OC)用于构建和分析全长、微量的cDNA(MQ-cDNA) 文库和用于表达分析的OC Unigene集合的开发。我们 WiFi利用我们当前的MQ-cDNA协议进行了许多细胞采购 具有代表性的永生化全长cDNA文库构建策略 从OC的细胞中分离，重点放在支持细胞上。以特定的目标 1、耳蜗毛细胞(Hcs)为 保证两种“永生化”的mq-cDNA模板的质量 和随后的mqcDNA文库。用于表达谱分析和细胞采购 我们将测试利用Chnra9-GFP转基因小鼠系的价值 在耳蜗毛细胞中表达绿色荧光蛋白。在特务目标2中， 将构建消减的归一化细胞特异性cDNA文库， 代表组织内的支持单元。这些是内部指骨， 内沟、内管、外管、Deiters的边缘细胞 亨森、S细胞以及与之接壤的克劳迪斯？细胞。细胞采购 协议，例如显微解剖、细胞采集，如果需要，还可以进行激光捕获 显微镜下，将与MQ-cDNA协议一起用于全长cDNA文库 制作。表达GFP的转基因小鼠将用于辅助隔离 OC支持小区的业务类型。对这些cDNA的质量评估将 通过在电子微阵列分析中使用来检测 离子通道基因在发育过程中表达，这是常见管家基因中罕见的 OC的基因、细胞特异性基因和仅在 非感觉/非神经细胞。在特定目标3中，细胞特异性克隆 取自AIMS 1和AIMS 2的芯片将被用来生成包含 OC“Unigene”套装。进一步识别新的和/或新的抄本 联合使用抑制性消减杂交和WIFI 微阵列分析。特定目标4将使用差异表达分析 使用OC芯片验证这些克隆的细胞特异性 结合零突变和自发突变的电子微阵列分析 老鼠。对表达谱的外推可以提供对 每种细胞类型在正常和正常状态下的功能特征和状态 OC的致病状态。