Conditional Inactivation of Synaptic proteins in transgenic mice

转基因小鼠突触蛋白的条件失活

• 批准号: 7637570
• 负责人: Craig C Garner
• 金额: $ 24.32万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-10 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7637570
• 关键词: Affect; Alzheimer' s Disease; Applications Grants; Axon; Behavior; Behavioral; Biochemical Genetics; Brain; Cell Culture Techniques; Cell membrane; Cells; Cellular Assay; Cognition Disorders; Collection; DNA; Data; Dementia; Development; Disease; Docking; Exons; Family; Figs - dietary; Future; Gene Family; Generations; Genes; Genetic; Genomics; Goals; Healthcare; Hippocampus (Brain); Incidence; Individual; Information Storage; Intellectual functioning disability; Knock-out; Knockout Mice; Knowledge; Label; Lentivirus Vector; Lesion; Maintenance; Molecular; Mus; Mutate; Nerve Degeneration; Neuraxis; Neuronal Differentiation; Neurons; Parkinson Disease; Phase; Play; Polymerase; Primary Cell Cultures; Process; Productivity; Protein Family; Protein Isoforms; Proteins; Publishing; Quality of life; RNA Interference; RNA Splicing; Reporter; Role; SNAP receptor; Scaffolding Protein; Schizophrenia; Signal Transduction; Site; Staging; Structural Protein; Structure; Subfamily lentivirinae; Synapses; Synaptic Cleft; Synaptic Vesicles; System; Techniques; Technology; Testing; Therapeutic; Time; Transgenes; Transgenic Mice; Transgenic Organisms; United States; Variant; Virus; age related; autism spectrum disorder; axonal pathfinding; base; cost; cost effective; depression; design; in vivo; information processing; innovation; knock-down; meetings; mouse model; neurodevelopment; neurotransmitter release; next generation; positional cloning; postsynaptic; presynaptic; promoter; protein complex; protein expression; protein function; public health relevance; red fluorescent protein; research study; small hairpin RNA; synaptic function; synaptogenesis; synaptotagmin; vector

DESCRIPTION (provided by applicant): Behavioral and cognitive disorders caused by genetic lesions, environmental insults or age related dementia are serious societal problems, contributing to a loss of quality of life for affected individuals and their families. The incidence of intellectual disability in the United States is 1-2%, costing tens of billions of dollars/year in health care and lost productivity. A key to defining therapeutic strategies that can ameliorate these disorders is a fundamental understanding of the cellular and molecular mechanisms regulating how neuronal circuits encode, process, and retain information. Synapses are the basic components of information storage and plasticity in our brains. Over the last decade, we have used molecular, cellular and reverse genetic approaches to identify and characterize proteins involved in the assembly, function and plasticity of vertebrate synapses. Increasingly, our data has shown that many of these proteins are transcribed from large multi-gene families spanning hundreds of kb of genomic DNA and comprised of multiple alternatively spliced exons. This complexity, as well as the cost and time associated with the generation of conditional knockout or knockin mice, has severely hampered progress in the field. What is needed is a simple, cost-effective strategy, akin to transgenics, for creating mice deficient in individual or combinations of proteins. Recent advances in transgenic technology using lentiviruses combined with interference RNAs are well poised to meet this challenge. Over the last five years, we have developed isoform specific short-hairpin RNAs (shRNAs) against numerous synaptic proteins as well as a collection of lentiviral vectors expressing XFP-tagged reporter proteins for the cell-autonomous and synapse specific analysis of pre and postsynaptic function. In the present application, we propose to integrate these technologies and the CRE/lox system to create an innovative set of lentiviruses capable of conditionally inactivating and expressing multiple neuronal proteins. Given their importance in the assembly and plasticity of synapses, we propose to use shRNAs against the structurally related presynaptic active zone proteins Piccolo and Bassoon to evaluate this new strategy. These conditional knockdown mice for Piccolo and/or Bassoon will be invaluable for assessing the shared and unique functions of these proteins during neuronal differentiation, axonal pathfinding, synapse formation, and in mechanisms of presynaptic plasticity at vertebrate synapses. PUBLIC HEALTH RELEVANCE: This grant application describes experiments designed to create a set of conditional transgenic knockdown mice deficient in the expression of the structurally related presynaptic active zone proteins Piccolo and Bassoon. In Aim 1, we propose to design, build and test the next generation of lentiviral vectors for the cost effective creation of conditional knockdown mice. Specifically, we will create a virus expressing three mini-genes: one an shRNA for Piccolo knockdown, the second a YFP-tagged Synapsin1a for labeling presynaptic boutons, and the third a red fluorescent protein variant (mCherry) for selecting mice with the integrated transgenes. The expression of each will be placed under the control of the CRE/lox system. The functionality of these vectors will be tested in HEK293 cells and cultured hippocampal neurons. In Aim 2, we will create and characterize a transgenic mouse using the lentiviral vector created in Aim1. In Aim 3, we will expand this technology to create a lentiviral vector capable of knocking down two or more synaptic proteins by expressing shRNAs for each protein under separate polymerase III promoters. This technology will be invaluable for studies of individual or families of proteins thought to perform similar functions. Moreover, it is ideally suited for a molecular replacement strategy that simultaneously eliminates the expression of one synaptic protein while replacing it with a mutated or altered version. Such a strategy could become crucial for the generation of mouse models of specific psychiatric, neurodegeneartive or neurodevelopment disorders such as depression, schizophrenia, or Autism Spectrum Disorders, or Alzheimer's disease.

描述（由申请人提供）：由遗传病变、环境损害或年龄相关痴呆引起的行为和认知障碍是严重的社会问题，导致患者及其家人的生活质量下降。在美国，智力残疾的发生率为1-2%，每年在医疗保健和生产力损失方面花费数百亿美元。定义可以改善这些疾病的治疗策略的关键是对调节神经元回路如何编码、处理和保留信息的细胞和分子机制的基本理解。突触是我们大脑中信息储存和可塑性的基本组成部分。在过去的十年中，我们已经使用分子，细胞和反向遗传方法来识别和表征与脊椎动物突触的组装，功能和可塑性有关的蛋白质。我们的数据越来越多地表明，许多这些蛋白质是从跨越数百kb基因组DNA的大型多基因家族转录而来，并由多个选择性剪接的外显子组成。这种复杂性，以及与产生条件敲除或敲入小鼠相关的成本和时间，严重阻碍了该领域的进展。我们所需要的是一种简单、经济的策略，类似于转基因，用于制造缺乏单个或组合蛋白质的小鼠。利用慢病毒与干扰rna结合的转基因技术的最新进展很好地应对了这一挑战。在过去的五年中，我们开发了针对许多突触蛋白的同种异型特异性短发夹rna (shRNAs)，以及一系列表达xfp标记的报告蛋白的慢病毒载体，用于突触前和突触后功能的细胞自主和突触特异性分析。在目前的应用中，我们建议将这些技术与CRE/lox系统相结合，以创建一套能够条件失活并表达多种神经元蛋白的创新慢病毒。鉴于shrna在突触组装和可塑性中的重要性，我们建议使用shrna对抗结构相关的突触前活性区蛋白Piccolo和Bassoon来评估这一新策略。这些短笛和/或巴松管的条件敲除小鼠对于评估这些蛋白质在神经元分化、轴突寻路、突触形成和脊椎动物突触突触前可塑性机制中的共同和独特功能将是非常宝贵的。公共卫生相关性：本拨款申请描述了旨在创建一组条件转基因敲低小鼠的实验，这些小鼠缺乏与结构相关的突触前活性区蛋白短笛和巴松管的表达。在目标1中，我们建议设计、构建和测试下一代慢病毒载体，以经济有效地创建条件敲低小鼠。具体来说，我们将创建一种表达三个迷你基因的病毒：一个是用于敲除短音的shRNA，第二个是用于标记突触前扣的yfp标记的Synapsin1a，第三个是用于选择整合转基因的小鼠的红色荧光蛋白变体（mCherry）。每个表达式都将置于CRE/lox系统的控制之下。这些载体的功能将在HEK293细胞和培养的海马神经元中进行测试。在Aim 2中，我们将使用在Aim1中创建的慢病毒载体创建和表征转基因小鼠。在Aim 3中，我们将扩展这项技术，创建一种慢病毒载体，能够通过在单独的聚合酶III启动子下表达每种蛋白质的shrna来敲低两种或多种突触蛋白。这项技术对于研究被认为具有类似功能的单个或家族的蛋白质将是无价的。此外，它非常适合于一种分子替代策略，即同时消除一种突触蛋白的表达，同时用突变或改变的版本代替它。这种策略对于产生特定精神疾病、神经退行性疾病或神经发育障碍（如抑郁症、精神分裂症、自闭症谱系障碍或阿尔茨海默病）的小鼠模型至关重要。