Models for Vision Research

描述（由申请人提供）：全世界约有5000万人失明，约1.5亿人视力严重受损。除了外伤和感染外，大多数人类眼病都是遗传性的。导致视网膜疾病的人类基因座的数量是可用的相关动物模型数量的约9倍，表明用于研究已知发生在人类中的疾病的模型存在很大差距。小鼠具有良好的遗传学，与人类生理学和解剖学的相似性，以及遗传操作的可访问性，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、用于整个发育和疾病进展的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。在上一个融资周期中，我们生成了超过60个具有眼部缺陷的模型。我们已经确定了20个突变株系的分子基础，从中获得了许多独特的见解。在本申请中，我们计划完成剩余40个品系（目标1）的分子和表型表征，并将其提供给科学研究界。扩展我们正在进行的遗传研究，我们建议使用敏化化学诱变筛选，以揭示重要的Crumbs 1途径（目的2）。虽然有许多策略可用于鉴定主要基因/突变的相互作用因子，但化学诱导突变的优势在于它们将允许无偏地鉴定与CRUMBS 1相互作用的广泛基因。这些基因可以解释与Crumbs 1内突变相关的大量疾病。它还将允许识别与CRUMBS 1的胞外结构域相互作用的因子，这是目前可用方法难以解决的一项奋进。在本申请中，我们将通过间接检眼镜检查筛选约10，000只诱变的G3 Crumbs 1 rd 8/rd 8小鼠，以鉴定存在改变的Crumbs 1 rd 8眼底表型的突变体。这些因素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CRUMBS 1途径的影响。该提案的成功结论不仅将产生良好表征的眼部模型，而且将潜在地确定CRUMBS 1途径的入口点以及在眼部生物学中重要的其他分子，并为我们提供建立和测试关于正常眼部功能和疾病病理学的假设的机会。 公共卫生相关性：几乎所有人类的非创伤性和非传染性眼病都是遗传性的，或者具有很强的遗传成分;遗传性疾病占常见眼病的95%以上。鉴定致病基因和动物模型以研究疾病进展和突变的病理后果是极其重要的。人类的许多眼部疾病，如果及早发现，可以治疗以减轻疾病过程。如果目前没有治疗方法，了解疾病的分子基础可以为新的治疗方案提供见解，然后可以使用模型来测试这些疗法。最后，了解致病基因及其作用途径可能会导致对确定预防视力损害和丧失的治疗靶点的理解。

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.