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、环境损伤或年龄相关性痴呆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庭生活质量下降。美国智力残疾的发病率为1- 2%，每年花费数百亿美元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存储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，细胞和反向遗传学的方法来确定和表征蛋白质参与的装配，功能和可塑性的脊椎动物突触。越来越多的数据表明，这些蛋白质中有许多是从跨越数百kb基因组DNA的大型多基因家族转录的，并由多个可变剪接外显子组成。这种复杂性，以及与产生条件性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。我们需要的是一种简单的、具有成本效益的策略，类似于转基因技术，用于制造缺乏单个蛋白质或蛋白质组合的小鼠。使用慢病毒结合干扰RNA的转基因技术的最新进展很好地准备迎接这一挑战。在过去的五年中，我们已经开发了针对许多突触蛋白的同种型特异性短发夹RNA（shRNA），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。在本申请中，我们提出整合这些技术和CRE/lox系统以创建一组能够条件性失活和表达多种神经元蛋白的创新慢病毒。鉴于它们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来评估这种新策略。这些条件敲低小鼠短笛和/或巴松管将是非常宝贵的，用于评估这些蛋白质的共享和独特的功能，在神经元分化，轴突寻路，突触形成，并在脊椎动物突触的突触前可塑性的机制。公共卫生关系：该授权申请描述了设计用于创建一组结构相关的突触前活性区蛋白Piccolo和Bassoon表达缺陷的条件性转基因敲除小鼠的实验。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性敲低小鼠。具体来说，我们将创建一种表达三个微型基因的病毒：一个是用于Piccolo敲低的shRNA，第二个是用于标记突触前终扣的YFP标记的Synapsin 1a，第三个是用于选择具有整合转基因的小鼠的红色荧光蛋白变体（mCherry）。每种基因的表达将置于CRE/lox系统的控制下。将在HEK 293细胞和培养的海马神经元中测试这些载体的功能。在目标2中，我们将使用Aim 1中创建的慢病毒载体创建并表征转基因小鼠。在目标3中，我们将扩展这项技术，以创建一种慢病毒载体，该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来敲低两种或更多种突触蛋白质。这项技术对于研究被认为具有类似功能的蛋白质个体或家族将是非常宝贵的。此外，它非常适合于分子替换策略，该策略同时消除一种突触蛋白的表达，同时用突变或改变的版本替换它。这种策略对于产生特定的精神病，神经退行性或神经发育障碍，如抑郁症，精神分裂症或自闭症谱系障碍或阿尔茨海默病的小鼠模型至关重要。