Mouse models for human deafness caused by diverse types of connexin26 mutations

描述（由申请方提供）：连接蛋白（Cx）是构成差距连接（GJ）的膜蛋白。GJ的主要功能是为细胞间的离子、电和生物化学（例如，营养素和信号分子）偶联。遗传学研究已经将Cx 26基因中的100多个突变与很大比例的语前非综合征性耳聋联系起来。尽管遗传研究揭示了GJ在听力中的重要性，但我们对它们在耳蜗中的功能作用知之甚少。一个主要原因是缺乏合适的动物模型，基于该模型可以研究由各种类型的Cx突变引起的人类遗传性耳聋。我们的长期目标是在分子水平上了解耳蜗GJ通道的功能。我们推测Cx 26的大截短突变和点突变的病理变化的分子机制是明显不同的。我们将使用基因工程技术来创建小鼠模型，这些模型在揭示耳蜗GJs的分子功能方面提供信息，并帮助我们了解各种类型的人类Cx 26突变引起的耳蜗病理。使用Cre-loxP介导的条件性基因靶向方法，我们将创建四个品系的小鼠模型，其代表如从体外研究中揭示的以下四种类型的人Cx 26突变：（1）导致生物化学通透性的选择性丧失的突变（例如，V84L）;（2）改变Ca++对Cx 26半通道的结合亲和力的突变，使得半通道在Ca++的生理浓度下渗漏（例如，G45 E）;（3）导致GJ全通道功能选择性丧失，但半通道功能基本不变的突变（例如，R75 W）;（4）GJ介导的细胞间通讯的完全丧失（例如，D179N）。然后，我们将检查四种转基因小鼠耳蜗的形态和功能变化。这些小鼠模型的可用性将极大地促进在分子水平上对最常见形式的先天性听力障碍的机制的调查。所获得的知识应该促进我们对GJ在耳蜗中的功能作用的理解，并有助于为大部分遗传性耳聋患者设计治疗策略。遗传学研究已经将连接蛋白基因中的100多个突变与大约一半的语前耳聋病例联系起来。本项目的重点是建立合适的动物模型，在此基础上研究由各种类型的Cx突变引起的人类遗传性耳聋。这些小鼠模型的可用性将极大地促进在分子水平上研究最常见形式的先天性听力障碍的机制。所获得的知识应该有助于我们为大部分遗传性耳聋患者设计更好的治疗策略。

DESCRIPTION (provided by applicant): Connexins (Cxs) are membrane proteins constituting the gap junctions (GJs). The major function of GJs is to provide a regulated conduit for intercellular ionic, electrical and biochemical (e.g., nutrients and signaling molecules) couplings. Genetic studies have linked more than 100 mutations in the Cx26 gene to a large proportion of prelingual nonsyndromic deafness. Despite the importance of GJs in hearing as revealed by genetic studies, we know very little about their functional role in the cochlea. One major reason is the lack of appropriate animal models based on which human hereditary deafness caused by various types of Cx mutations could be studied. Our long-term goal is to understand the function of GJ channels in the cochlea at the molecular level. We hypothesize that the molecular mechanisms underlying pathological changes of large truncation mutations and point mutations of Cx26 are markedly different. We will use genetic engineering techniques to create mouse models that are both informative in revealing the molecular function of cochlear GJs and in helping us understand cochlear pathologies caused by various types of human Cx26 mutations. Using the Cre-loxP mediated conditional gene targeting method, we will create four lines of mouse models representing the following four types of human Cx26 mutations as revealed from in vitro studies: (1) mutations that cause a selective loss of biochemical permeability (e.g., V84L); (2) mutations that alter the Ca++ binding affinity to Cx26 hemichannels, such that hemichannels are leaky at physiological concentrations of Ca++ (e.g., G45E); (3) mutations that cause a selective loss of GJ whole channel functions, but the hemichannel function is largely unchanged (e.g., R75W); (4) total loss of GJ- mediated intercellular communications (e.g., D179N). We will then examine the morphological and functional changes in the cochleae of the four lines of genetically modified mice. The availability of these mouse models will greatly facilitate investigations into the mechanism of the most common form of congenital hearing impairment at the molecular level. The knowledge gained should advance our understanding of the functional roles of GJs in the cochlea as well as help designing treatment strategies for a large portion of hereditary deafness patients. Genetic studies have linked more than 100 mutations in the connexin genes to about half of all cases of prelingual deafness. The focus of this project is to establish appropriate animal models based on which human hereditary deafness caused by various types of Cx mutations could be studied. The availability of these mouse models will greatly facilitate investigations into the mechanism of most common form of congenital hearing impairment at the molecular level. The knowledge gained should help us design better treatment strategies for a large portion of hereditary deafness patients.