Identification and Functional Analysis of the Mouse Deaf

聋鼠的鉴定及功能分析

• 批准号: 6634511
• 负责人: David C Kohrman
• 金额: $ 23.49万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6634511
• 关键词: cochlea; deafness; electrophysiology; gene expression; gene mutation; genetic mapping; genetically modified animals; human genetic material tag; laboratory mouse; molecular cloning; nucleic acid sequence; protein localization; sensorineural hearing loss

DESCRIPTION: (Adapted from applicant's abstract): The primary objective of this proposal is the identification and characterization of the gene responsible for inherited deafness in the mouse mutant known as 'spinner'. Mutation of this gene results in sensorineural hearing loss similar to that found in many human nonsyndromic deafness disorders. The approaches described will attempt to correlate effects at the single gene level with those occurring at the cellular level and the level of the intact cochlea. A positional cloning strategy will be used to identify the affected gene. Gene localization information will be derived from an existing high resolution genetic cross and a set of genomic DNA clones that span the candidate region. Positional candidate genes mapped to this region in mouse, and to the region of conserved linkage in humans, will be evaluated for mutation in spinner mice. To further narrow the candidate region, genomic DNA clones from the candidate region will be microinjected into sr/sr zygotes. DNA clones that contain a functional version of the normal gene are expected to produce phenotypic correction in the resulting transgenic progeny. This DNA will be directly screened for the presence of the affected gene. The biological role of the spinner gene in the cochlea will be examined using several approaches. High resolution phenotypic analysis of affected mice will be performed to identify early defects in the cochlea that result from mutation of the spinner gene. Sensory cell function in the cochlea will be assessed by measurement of evoked responses in spinner mice. Ultrastructural and immunocytochemical analyses will be performed to detail the degenerative process in the mutant cochlea. Database analysis of the gene's primary sequence will be used to identify related genes and protein motifs that may provide insight into gene function. The expression pattern of the spinner gene, and the subcellular localization of its encoded protein, will be determined. Based upon comparative genetic data, the human version of the spinner gene is a positional candidate for the gene affected in a nonsyndromic deafness disorder, DFNB6. The human gene will be directly evaluated for mutations in individuals with inherited defects at the DFNB6 locus. This project will result in the identification of a critical gene in the mammalian inner ear, provide the basis for a model of this gene's role in the cochlea, and investigate the involvement of the gene in human nonsyndromic hearing loss.

描述：（改编自申请人的摘要）：此项目的主要目标 提案是负责基因的识别和表征 被称为“旋转者”的小鼠突变体遗传性耳聋。这个突变 基因导致的感音神经性听力损失与许多人类中发现的相似 非综合征性耳聋疾病。所描述的方法将尝试 将单基因水平的影响与细胞中发生的影响相关联 水平和完整耳蜗的水平。定位克隆策略将 用于识别受影响的基因。基因定位信息将 源自现有的高分辨率遗传杂交和一组基因组 DNA 跨越候选区域的克隆。位置候选基因映射到 小鼠中的该区域以及人类中的保守连接区域将被 评估旋转小鼠的突变。为了进一步缩小候选区域， 来自候选区域的基因组 DNA 克隆将被显微注射到 sr/sr 中 受精卵。含有正常基因功能版本的 DNA 克隆是 预计将在所得转基因后代中产生表型校正。 将直接筛选该 DNA 中是否存在受影响的基因。这 旋转基因在耳蜗中的生物学作用将使用以下方法进行检查 几种方法。对受影响小鼠进行高分辨率表型分析将 进行以确定由突变引起的耳蜗早期缺陷 的旋转基因。耳蜗中的感觉细胞功能将通过以下方式进行评估 旋转小鼠诱发反应的测量。超微结构和 将进行免疫细胞化学分析以详细说明退行性病变 突变耳蜗中的过程。基因一级序列的数据库分析 将用于识别可能提供的相关基因和蛋白质基序 深入了解基因功能。 spinner 基因的表达模式 其编码蛋白的亚细胞定位将被确定。基于 比较遗传数据，人类版本的旋转基因是一个位置 受非综合征性耳聋疾病影响的候选基因 DFNB6。这 将直接评估患有以下疾病的个体的人类基因突变 DFNB6 位点的遗传缺陷。该项目将导致 鉴定哺乳动物内耳中的关键基因，提供基础 建立该基因在耳蜗中的作用模型，并研究其参与情况 人类非综合征性听力损失的基因。