Identification and Functional Analysis of the Mouse Deaf

描述：(改编自申请者摘要)：本报告的主要目的 建议是对负责的基因进行鉴定和表征 遗传性耳聋的小鼠突变体被称为“旋转者”。这是一个突变 基因导致的感音神经性听力损失与许多人相似 非综合征性耳聋。所描述的方法将尝试 单基因水平上的效应与细胞内发生的效应相关 水平和完整耳蜗处的水平。位置克隆策略将 被用来鉴定受影响的基因。基因定位信息将被 来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。定位候选基因被定位到 小鼠的这一区域，以及人类的保守连锁区域，将是 对旋转小鼠的突变进行评估。为了进一步缩小候选区域， 候选区域的基因组DNA克隆将被显微注射到锶/锶中 受精卵。包含正常基因功能版本的DNA克隆是 预计将在由此产生的转基因后代中产生表型纠正。 该DNA将被直接筛选，以确定是否存在受影响的基因。这个 旋转器基因在耳蜗中的生物学作用将通过 有几种方法。受影响小鼠的高分辨率表型分析 以确定因突变而导致的耳蜗骨早期缺陷 旋转者的基因。耳蜗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变 突变型耳蜗内的突起。该基因一级序列的数据库分析 将用于识别相关基因和蛋白质基序，这些基因和蛋白质基序可能提供 对基因功能的洞察。旋转器基因的表达模式，以及 其编码蛋白的亚细胞定位将被确定。基于 比较遗传数据，人类版本的旋转器基因是一个位置 一种非综合征性耳聋的候选基因DFNB6。这个 人类基因将被直接评估为患有 DFNB6基因座的遗传性缺陷。这一项目将导致 鉴定哺乳动物内耳中的一个关键基因，为进一步研究提供依据 寻找该基因在耳蜗中作用的模型，并研究其参与 该基因在人类非综合征性听力损失中的作用。

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.