Transgenic and knockout approaches to study protocadherin function

研究原钙粘蛋白功能的转基因和敲除方法

基本信息

批准号：
8547843
负责人：
JAMES DAVID JONTES
金额：
$ 18.3万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-19 至 2015-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8547843
关键词：
Autistic Disorder Automobile Driving Behavior Biological Models Brain Brain Diseases Brain region Cadherins Cell Proliferation Cell Surface Receptors Cell physiology Cells Collection Complex Data Defect Development Developmental Process Disease Distributed Systems Embryo Enhancers Epilepsy Etiology Family Female Foundations Functional disorder Gene Transfer Techniques Genes Genetic Genome Germ-Line Mutation Goals Health Human Image Incidence Infant Knock-out Knowledge Label Larva Lead Lesion Light Mediating Mental Retardation Microscopy Modeling Molecular Mutation Nervous system structure Neurites Neurodevelopmental Disorder Neurons Neurosciences Pattern Phenotype Photons Positioning Attribute Process Reagent Regulatory Element Retina Role Schizophrenia Stereotyping Surveys Technology Testing Time Transgenes Transgenic Organisms Visual system structure Work Zebrafish Zinc Fingers autism spectrum disorder axon guidance base cell motility in vivo insight migration mutant nervous system development neural circuit neurodevelopment neuronal cell body neuronal patterning nuclease promoter relating to nervous system self assembly superior colliculus Corpora quadrigemina synaptogenesis

项目摘要

DESCRIPTION (provided by applicant): The development of the vertebrate brain requires complex genetic networks to coordinate myriad processes across time and space, which include cell proliferation, differentiation and migration, along with axon guidance, synapse formation and elimination. Orchestration of these processes results in the assembly of microcircuitry and distributed systems that constitute a functional nervous system. Disruptions in neural development can have a dramatic impact, resulting in complex brain disorders, such as autism spectrum disorders, schizophrenia, mental retardation and epilepsy. Increasingly, genetic data implicate genes involved in axon guidance and synaptogenesis in these complex brain disorders. In addition, evidence suggests a common etiology for these disorders, as mutations associated with one disorder are also associated with increased incidence of others. Among the molecules that have been implicated in these complex brain disorders are the protocadherins. In particular, mutations in pcdh19 result in a female-limited form of infant-onset epilepsy. Despite the clear role for these molecules in neural development and the etiology of neurodevelopmental dysfunction, relatively little is known about their function in vivo. Here, we propose to generate lines of zebrafish in which pcdh7a, pcdh9, pcdh17 and pcdh19 have been inactivated using zinc finger nucleases (ZFNs). In addition, we propose to generate BAC transgenic lines expressing GFP under the control of the pcdh7a, pcdh9, pcdh17 and pcdh19 regulatory elements. To study the effects of the protocadherin mutations on neural development, we will cross the GFP lines into the mutant backgrounds and use in vivo 2-photon microscopy to determine the impact of the mutations on nervous system development. This work will significantly advance our understanding of protocadherin function, providing a foundation for more mechanistic studies and facilitating a better understanding of how disruption of protocadherins can lead to complex brain disorders.

描述（由申请人提供）：脊椎动物大脑的发展需要复杂的遗传网络来协调跨时间和空间的众多过程，其中包括细胞增殖，分化和迁移，以及轴突指导，突触形成和消除。这些过程的编排导致构成功能性神经系统的微电路和分布式系统组装。神经发育的破坏会产生巨大的影响，从而导致复杂的脑部疾病，例如自闭症谱系障碍，精神分裂症，智力低下和癫痫。越来越多的遗传数据暗示了这些复杂脑部疾病中参与轴突引导和突触发生的基因。此外，有证据表明，这些疾病的常见病因学，因为与一种疾病相关的突变也与其他疾病的发病率增加有关。在这些复杂的脑部疾病中涉及的分子中，原核蛋白是原始蛋白。特别是，PCDH19中的突变导致女性限制的婴儿发作癫痫。尽管这些分子在神经发育和神经发育功能障碍的病因中起着明显的作用，但对它们在体内的功能知之甚少。在这里，我们建议生成斑马鱼线，其中PCDH7A，PCDH9，PCDH17和PCDH19已使用锌指核酸酶（ZFN）灭活。此外，我们建议在PCDH7A，PCDH9，PCDH17和PCDH19调节元件的控制下产生表达GFP的BAC转基因线。为了研究原钙粘着蛋白突变对神经发育的影响，我们将跨越GFP线到突变背景中，并在体内2-光子显微镜中使用以确定突变对神经系统发育的影响。这项工作将大大提高我们对原钙粘蛋白功能的理解，为更多机械研究提供基础，并促进对原始蛋白的破坏如何导致复杂的脑部疾病的更好理解。