A conditional RNAi approach to find genes required for hearing

描述(申请人提供)：声音由内耳的毛细胞和神经元检测和编码，并由中枢听觉系统的复杂电路处理。数以百万计的人在对声音的检测或感知方面经历了一定程度的听力障碍，从深度耳聋到耳鸣和学习障碍。听力研究的一个主要目标是找到听力所需的基因，以改进对这种广泛的外周和中枢性听力障碍的诊断和治疗。在模式生物中的基因筛选，以及对人类耳聋基因的定位克隆，已经发现了许多耳聋基因，并极大地促进了我们对听觉系统如何工作的理解。然而，这种认识仍然不完整，因为机械转导通道尚未确定这一事实突出了这一点。此外，由于诊断工具不足以及缺乏正常听觉回路组装和功能的知识，人们对中枢听觉处理障碍的患病率或病因学知之甚少。这项研究的长期目标是开发一种新的基因发现方法，以补充正在进行的耳聋基因筛查，并扩大我们对听力分子基础的理解。我们建议创造一种在体内快速干扰听觉基因功能的新方法。这项技术将在小鼠体内使用Cre-lox技术和RNA干扰(RNAi)来扰乱内耳受限细胞群体中假定的耳聋基因的活性。ShRNA的产生将与一种荧光标记的激活有关，从而可以轻松地显示下至突触水平的神经元形态。该方法依赖于携带两组不相容的Cre识别位点的转基因，即U6启动子、CAG启动子以及DsRed和Venus编码序列。这些元件被配置成使Cre介导的重组导致基因特异性shRNA的表达，并同时从红色荧光切换到黄色荧光。转基因将定位于胚胎干细胞中的一个特定位点，用于建立RNAi小鼠品系。然后，这些小鼠可以与内耳特异的Cre驱动程序杂交，从而避开多能性效应和致命性。第一个目标是创建一种在体外有效工作的Cre-RNAi载体。第二个目的是通过靶向在HDR综合征中突变的已知耳聋基因GATA3来验证该载体与听觉系统体内筛查的兼容性。为此，我们将比较GATA3-RNAi和传统的条件性GATA3基因敲除小鼠的表型，利用基因敲除小鼠的金星荧光来可视化耳蜗线的变化。除了提供一种寻找听力所需基因的新工具外，这项技术还可以扩展，以产生靶向ES细胞资源，这些细胞可以在神经系统的任何区域进行功能筛选。 公共卫生相关性：数百万人经历某种形式的听力障碍，从深度耳聋到导致耳鸣和学习障碍的中枢听觉处理障碍。识别听力所必需的基因将改善对一系列疾病的诊断和治疗。这个项目的目标是开发一种新的方法来探测老鼠的基因功能，这将促进我们对听觉系统如何正常运作以及当它在人类身上不起作用时会发生什么的理解。

DESCRIPTION (provided by applicant): Sound is detected and encoded by hair cells and neurons in the inner ear and processed by complex circuits in the central auditory system. Millions of people experience a degree of hearing impairment in either the detection or perception of sound, ranging from profound deafness to tinnitus and learning disabilities. A major objective in hearing research is to find the genes required for hearing in order to improve the diagnosis and treatment of this wide array of peripheral and central auditory disorders. Genetic screens in model organisms, together with positional cloning of human deafness loci, have uncovered many deafness genes and have greatly advanced our understanding of how the auditory system works. However, this knowledge remains incomplete, as highlighted by the fact that the mechanotransduction channel has yet to be identified. Moreover, little is known about the prevalence or etiology of central auditory processing disorders, due to inadequate diagnostic tools and a lack of knowledge of normal auditory circuit assembly and function. The long-term goal of this study is to develop a new method of gene discovery that will complement ongoing screens for deafness genes and expand our understanding of the molecular basis of hearing. We propose to create a new method for rapidly disrupting auditory gene function in vivo. This technique will use Cre-lox technology and RNA interference (RNAi) in the mouse to disrupt the activity of putative deafness genes in restricted cell populations of the inner ear. shRNA production will be linked to activation of a fluorescent marker, permitting easy visualization of neuronal morphology down to the level of the synapse. The method relies on a transgene that carries two sets of incompatible Cre recognition sites, a U6 promoter, a CAG promoter, and the DsRed and Venus coding sequences. These elements are configured such that Cre-mediated recombination results in expression of a gene-specific shRNA and a simultaneous switch from red to yellow fluorescence. The transgene will be targeted to a defined locus in embryonic stem cells, which will be used to establish lines of RNAi mice. These mice can then be crossed to inner ear-specific Cre drivers, circumventing pluripotent effects and lethality. The first aim is to create a Cre-RNAi vector that works effectively in vitro. The second aim is to validate the compatibility of this vector with an in vivo screen in the auditory system by targeting a known deafness gene, GATA3, which is mutated in HDR syndrome. To this end, we will compare the phenotypes of Gata3-RNAi and conventional conditional Gata3 knockout mice, taking advantage of the Venus fluorescence in knockdown mice to visualize changes in cochlear wiring. As well as providing a novel tool for finding genes required for hearing, this technique can be expanded to generate a resource of targeted ES cells that can be screened for function in any region of the nervous system. PUBLIC HEALTH RELEVANCE: Millions of people experience some form of hearing impairment, from profound deafness to central auditory processing disorders that contribute to tinnitus and learning disabilities. The identification of genes necessary for hearing will improve the diagnosis and treatment of a wide array of disorders. The goal of this project is to develop a new method of probing gene function in mice that will advance our understanding of how the auditory system normally functions and what happens when it doesn't in humans.