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

• 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
• 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

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）：脊椎动物脑的发育需要复杂的遗传网络来协调跨越时间和空间的无数过程，其包括细胞增殖、分化和迁移，以及沿着轴突引导、突触形成和消除。这些过程的演示导致微电路和分布式系统的组装，构成功能神经系统。神经发育的中断会产生巨大的影响，导致复杂的大脑疾病，如自闭症谱系障碍、精神分裂症、智力迟钝和癫痫。越来越多的遗传数据表明，在这些复杂的大脑疾病中，基因参与了轴突导向和突触发生。此外，有证据表明这些疾病的共同病因，因为与一种疾病相关的突变也与其他疾病的发病率增加相关。在这些复杂的大脑疾病中涉及的分子是原钙粘蛋白。特别是，pcdh19基因突变导致女性局限性的婴儿癫痫。尽管这些分子在神经发育和神经发育功能障碍的病因学中具有明确的作用，但对其在体内的功能知之甚少。在这里，我们建议产生线的斑马鱼，其中pcdh7a，pcdh9，pcdh17和pcdh19已被灭活使用锌指核酸酶（ZFN）。此外，我们建议产生BAC转基因株系，表达GFP的控制下的pcdh7a，pcdh9，pcdh17和pcdh19调节元件。为了研究原钙粘蛋白突变对神经发育的影响，我们将GFP细胞系交叉到突变背景中，并使用体内双光子显微镜来确定突变对神经系统发育的影响。这项工作将大大推进我们对原钙粘蛋白功能的理解，为更多的机制研究提供基础，并促进更好地了解原钙粘蛋白的破坏如何导致复杂的大脑疾病。