Models for Vision Research

描述(申请人提供)：全球约有5000万人失明，约1.5亿人视力严重受损。除了创伤和感染，大多数人类眼部疾病本质上都是遗传的。导致视网膜疾病的人类基因座数量是可用的相关动物模型数量的~9倍，这表明在研究已知发生在人类身上的疾病的模型方面存在很大差距。小鼠由于其发达的遗传学、与人类生理和解剖学的相似性以及遗传操作的可及性，是一种被广泛接受和有用的模型系统。小鼠模型已被用于提供人类疾病的候选基因，用于在整个发育和疾病进展过程中进行研究的组织，以及用于治疗的测试系统。它们也是通过遗传手段识别和剖析生物相关途径的理想平台。在上一个资助周期中，我们制作了60个眼睛缺陷的模型。我们已经确定了20个突变系的分子基础，从这些突变系中我们获得了许多独特的见解。在这项应用中，我们计划完成剩余的40个品系的分子和表型特征(目标1)，并将它们提供给科学研究界。扩展我们正在进行的遗传学研究，我们建议使用敏化的化学诱变筛选来揭示在Crums1途径中重要的途径(目标2)。虽然有许多策略可用于识别主要基因/突变的相互作用因素，但化学诱导突变的优势在于，它们将允许无偏见地识别与CRUMBS1相互作用的广泛基因。这些基因可能解释了与Crums1基因突变相关的过多疾病。它还将允许识别与CRUMBS1的胞外结构域相互作用的因素，这是目前可用的方法难以完成的一项工作。在本应用中，我们将通过间接眼底镜筛选约10,000只突变的G3Crums1rd8/Rd8小鼠，以确定存在Crums1drd8眼底表型改变的突变。这些因子的分子基础将被确定，并通过使用标准的免疫组织化学方法结合使用4PI显微镜，我们将检查新发现的基因/突变对CRUMBS1途径的影响。这一提议的成功完成不仅将产生具有良好特征的眼模型，还将潜在地确定CRUMBS1通路以及其他在眼生物学中重要的分子的切入点，并为我们提供机会建立和测试关于正常眼功能和疾病病理学的假说。 与公共卫生相关：人类几乎所有的非外伤性和非传染性眼部疾病都是遗传起源的或具有很强的遗传成分；遗传性疾病占常见眼病的95%以上。识别致病基因和动物模型来研究疾病的进展和突变的病理后果是极其重要的。人类的许多眼病，如果及早发现，可能会得到治疗，以减缓疾病的进程。如果目前还没有可用的治疗方法，了解这种疾病的分子基础可能会为新的治疗方案提供见解，然后这些模型可以用于测试这些疗法。最后，对致病基因及其功能途径的了解可能会导致对确定预防视力损害和丧失的治疗目标至关重要的理解。

DESCRIPTION (provided by applicant): Approximately 50 million people worldwide are blind and ~150 million are significantly vision impaired. Except for trauma and infections, the majority of human eye diseases are genetic in nature. The number of human loci causing retinal disease is ~ 9-fold greater than the number of available associated animal models, indicating a large gap in models for studying diseases that are known to occur in humans. The mouse with its well-developed genetics, similarity to human physiology and anatomy, and accessibility for genetic manipulation is a widely accepted and useful model system. Mouse models have been used to provide candidate genes for human diseases, tissues for study throughout development and disease progression, and test systems for therapies. They are also an ideal platform to identify and dissect biologically relevant pathways through genetic means. In the last funding cycle, we generated >60 models with ocular defects. We have identified the molecular basis of 20 of the mutant lines from which many unique insights were obtained. In this application, we plan to complete the molecular and phenotypic characterization of the 40 remaining lines (Aim 1) and make them available to the scientific research community. Extending our ongoing genetic studies, we propose to use a sensitized chemical mutagenesis screen to reveal pathways important in the Crumbs1 pathway (Aim 2). While there are many strategies available to identify interacting factors of primary genes/mutations, chemically induced mutations have the advantage that they will allow for the unbiased identification of a wide array of genes that interact with CRUMBS1. These genes may explain the plethora of diseases associated with mutations within Crumbs1. It will also allow for identification of factors that interact with the extracellular domain of CRUMBS1, an endeavor that has been intractable by the current available methods. In the present application, we will screen ~10,000 mutagenized G3 Crumbs1rd8/rd8 mice by indirect ophthalmoscopy to identify mutants that present with an altered Crumbs1drd8 fundus phenotype. The molecular bases of these factors will be identified and through the use of standard immunohistochemical methodologies in conjunction with the use of 4Pi microscopy, we will examine the effects of the newly identified genes/mutations on the CRUMBS1 pathway. Successful conclusion of this proposal will not only generate well characterized ocular models, but will potentially identify entry points into the CRUMBS1 pathways as well as other molecules that are important in eye biology and afford us the opportunity to build and test hypotheses about normal ocular function and disease pathology. PUBLIC HEALTH RELEVANCE: Virtually all non-traumatic and non-infectious ocular diseases in humans are genetic in origin or have a strong genetic component; genetic disorders account for more than 95% of common eye diseases. Identification of disease causing genes and animal models to study the disease progression and pathological consequences of mutations is extremely important. Many eye diseases in humans, if identified early enough, may be treated to attenuate the disease process. If no treatment is currently available, knowing the molecular basis of the disease may provide insights to new treatment regimens and the models can then be used to test those therapeutics. Finally, knowledge of the disease causing genes and the pathways in which they function may lead to an understanding that is critical in defining therapeutic targets for prevention of vision impairment and loss.