Identification and Functional Analysis of the Mouse Deaf

描述：（改编自申请人摘要）：本研究的主要目的 建议是鉴定和表征基因负责 遗传性耳聋的老鼠突变体被称为“纺纱工”。这种突变 基因导致的感觉神经性听力损失与许多人类 非综合征性耳聋所描述的方法将试图 将单基因水平的效应与细胞水平的效应相关联， 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。定位候选基因映射到 小鼠中的该区域以及人类中的保守连锁区域将被 在旋转小鼠中评估突变。为了进一步缩小候选区域， 将来自候选区域的基因组DNA克隆显微注射到sr/sr中， 受精卵含有正常基因的功能性版本的DNA克隆， 预期在所得转基因后代中产生表型校正。 该DNA将被直接筛查受影响基因的存在。的 spinner基因在耳蜗中的生物学作用将用 几种方法。受影响小鼠的高分辨率表型分析将 以识别由突变引起的耳蜗早期缺陷 spinner基因将通过以下方法评估耳蜗中的感觉细胞功能： 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 变异耳蜗的突起基因一级序列数据库分析 将用于识别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， 将确定其编码蛋白的亚细胞定位。基于 比较遗传数据，人类版本的spinner基因是一个位置， 在非综合征性耳聋疾病中受影响的基因DFNB 6的候选者。的 人类基因将被直接评估， DFNB 6基因座的遗传缺陷该项目将导致 哺乳动物内耳中一个关键基因的鉴定， 为该基因在耳蜗中的作用建立模型，并调查其参与程度。 人类非综合征性听力损失的基因

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.