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: 7802809
• 负责人: Craig C Garner
• 金额: $ 20.11万
• 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/7802809
• 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; Mental Depression; 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; 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，环境侮辱或与年龄相关的痴呆症引起的行为和认知障碍是严重的社会问题，导致受影响的个人及其家人的生活质量丧失。美国智力残疾的发生率为1-2％，损失每年的医疗保健和生产力损失。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电路如何编码，过程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存储和可塑性的基本组成部分。在过去的十年中，我们使用了分子，细胞和反向遗传学方法来识别和表征与脊椎动物突触组装，功能和可塑性有关的蛋白质。我们的数据越来越多地表明，这些蛋白质中的许多蛋白质是从跨越数百种基因组DNA的大型多基因家族中转录的，由多个剪接的外显子组成。这种复杂性，以及与有条件敲除或敲除小鼠产生的成本和时间，严重阻碍了该领域的进步。需要的是一种简单，具有成本效益的策略，类似于转基因，用于创建缺乏个体或蛋白质组合的小鼠。使用慢病毒和干扰RNA结合使用的转基因技术的最新进展已很好地应对这一挑战。在过去的五年中，我们开发了针对众多突触蛋白的同工型特异性短发（SHRNA），以及表达XFP标记的报道蛋白的慢病毒载体的集合，用于细胞自主和突触的突触特异性分析前和突触后功能。在本应用程序中，我们建议将这些技术和CRE/LOX系统整合起来，以创建一组创新的烟病毒，能够有条件地失活和表达多种神经元蛋白。鉴于它们在突触的组装和可塑性中的重要性，我们建议使用SHRNA来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。这些有条件的Piccolo和/或Bassoon的有条件的敲低小鼠对于评估这些蛋白质在神经元分化，轴突探路，突触形成和椎骨突触时突触前可塑性机制中的共同和独特功能是无价的。公共卫生相关性：该赠款应用程序描述了旨在创建一组有条件的转基因敲低小鼠的实验。在AIM 1中，我们建议设计，构建和测试下一代的慢病毒向量，以有效地创建有条件的敲低小鼠。具体而言，我们将创建一种表达三个小型生成的病毒：一种用于Piccolo敲低的shRNA，第二种是YFP标记的Synapsin1a，用于标记突触前胸子，第三个是红色荧光蛋白变体（MCHERRY），用于选择具有集成转移的小鼠。每个的表达将放置在CRE/LOX系统的控制下。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AIM1中创建的慢病毒向量创建和表征转基因鼠标。在AIM 3中，我们将扩展这项技术，以创建一个慢病毒载体，能够通过在单独的聚合酶III启动子下为每种蛋白质表达shRNA来击倒两种或多种突触蛋白。这项技术对于认为具有类似功能的个体或蛋白质家族将是无价的。此外，它非常适合一种分子替代策略，该策略同时消除了一种突触蛋白的表达，同时用突变或变化的版本代替它。这种策略对于生成特定精神病，神经变性或神经发育障碍的小鼠模型（例如抑郁症，精神分裂症或自闭症谱系障碍或阿尔茨海默氏病）至关重要。