Transcriptome and Proteome Analysis of Mouse Inner Ears

小鼠内耳的转录组和蛋白质组分析

• 批准号: 7030056
• 负责人: QING Y ZHENG
• 金额: $ 15.75万
• 依托单位: JACKSON LABORATORY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-19 至 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7030056
• 关键词: cell morphology; ear hair cell; gene expression profiling; genetic models; hearing disorders; immunocytochemistry; in situ hybridization; laboratory mouse; labyrinth; mass spectrometry; microarray technology; molecular biology information system; molecular pathology; organ of Corti; polymerase chain reaction; proteomics; two dimensional gel electrophoresis

DESCRIPTION (provided by applicant): The broad long-term objective of this proposed project is to uncover the pathophysiological mechanisms of human auditory diseases by analyzing mouse genetic models using genomic and proteomic approaches. Few protein-profiling studies of mouse ear tissues have been attempted because of the difficulty of accessing the small numbers of diverse cells within the hard temporal bone that encases the inner ear. To address this problem, a subtractive strategy was devised in which the digitized abundance of transcripts and proteins in mutant mice with lost inner ear structures is subtracted from those of control mice with normal inner structures. For example, a predominate histological feature of the ears of mice homozygous for the dreidel (ddl) mutation of Pou4f3 is a lack of hair cells in the organ of Corti (OC). Therefore transcripts and proteins of hair cells will be revealed by subtraction of the OC contents of ddl/ddl mutants from those of control mice. The subtractive digitized comparisons of proteome content will be performed using the newly developed two-dimensional difference in gel electrophoresis (2D DICE) method for protein separation and mass spectrometry (MS) for protein identification. Transcript comparisons between mutants and controls will be made by cDNA microarray analysis. Immunohistochemistry and real time RT- PCR approaches will be used to validate protein and mRNA expression patterns and confirm inner ear localization of genes that are identified by 2D DIGE and MS. In this way, a catalog of transcriptome and proteome contents of particular inner ear structures will be compiled for mice at different ages, and an online database will be established to execute a data-sharing plan. This proposal is in response to the NIH Program Announcement PA-03-151 "Proteomics in Auditory Developmental and Disease Processes." The relevance of the proposed research to public health: Techniques developed and data to be collected in this project will be useful for developing strategies for prevention and treatment of human ear diseases. Genes, proteins and their molecular pathways identified in this proposed research are potential targets of drug therapies for hearing loss that affects more than 28 million Americans.

描述（由申请人提供）：该提出的项目的广泛长期目标是通过使用基因组和蛋白质组学方法分析小鼠遗传模型来揭示人类听觉疾病的病理生理机制。由于难以访问包围内耳的硬颞骨中的少量细胞，因此很少尝试对小鼠耳组织进行蛋白质促进研究。为了解决这个问题，设计了一种减法策略，其中从具有正常内部结构的对照小鼠的内耳小鼠中减去了突变小鼠中数字化的成绩单和蛋白质。例如，POU4F3的DREIDEL（DDL）突变的小鼠耳朵的主要组织学特征是Corti（OC）的器官中缺乏毛细胞。因此，毛细胞的转录本和蛋白质将通过从对照小鼠的OC含量减去DDL/DDL突变体的OC含量来揭示。将使用新开发的凝胶电泳二维差异（2D骰子）进行蛋白质分离和质谱法（MS）的新开发的二维差异来进行蛋白质组含量的减法化比较。突变体和对照组之间的转录本比较将通过cDNA微阵列分析进行。免疫组织化学和实时RT-PCR方法将用于验证蛋白质和mRNA表达模式，并确认由2D DIGE和MS鉴定的基因的内耳定位。这样，将为不同年龄的小鼠编译特定内耳结构的转录组和蛋白质组内容目录，并将建立一个在线数据库来执行数据共享计划。该建议是对NIH计划公告PA-03-151“听觉发育和疾病过程中的蛋白质组学”的回应。 拟议的研究与公共卫生的相关性：开发的技术和在本项目中收​​集的数据将有助于制定预防和治疗人耳部疾病的策略。在这项拟议的研究中鉴定出的基因，蛋白质及其分子途径是影响超过2800万美国人的听力损失的潜在靶标。