Conditional Inactivation of Synaptic proteins in transgenic mice

转基因小鼠突触蛋白的条件失活

基本信息

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
项目类别：
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&apos 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

项目摘要

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）：由遗传损伤、环境损害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，导致受影响个人及其家庭的生活质量下降。在美国，智力障碍的发生率为 1-2%，每年造成数百亿美元的医疗费用和生产力损失。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了解。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，我们使用分子、细胞和反向遗传方法来识别和表征参与脊椎动物突触组装、功能和可塑性的蛋白质。我们的数据越来越多地表明，其中许多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。这种复杂性以及与条件敲除或基因敲入小鼠的产生相关的成本和时间，严重阻碍了该领域的进展。我们需要的是一种简单、经济有效的策略，类似于转基因技术，用于培育缺乏单个蛋白质或蛋白质组合的小鼠。使用慢病毒与干扰 RNA 相结合的转基因技术的最新进展已做好应对这一挑战的准备。在过去五年中，我们开发了针对多种突触蛋白的亚型特异性短发夹 RNA (shRNA)，以及一系列表达 XFP 标记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，我们建议将这些技术和 CRE/lox 系统整合起来，创建一套创新的慢病毒，能够有条件地灭活和表达多种神经元蛋白。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 针对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、突触形成以及脊椎动物突触突触前可塑性机制过程中的共享和独特功能将具有无价的价值。公共健康相关性：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，以经济高效地创建条件敲除小鼠。具体来说，我们将创建一种表达三个小基因的病毒：一个是用于 Piccolo 敲低的 shRNA，第二个是用于标记突触前 bouton 的 YFP 标记的 Synapsin1a，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) 。每个的表达都将置于 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。在目标 2 中，我们将使用目标 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，创建一种慢病毒载体，通过在单独的聚合酶 III 启动子下表达每种蛋白质的 shRNA，能够敲除两种或多种突触蛋白质。这项技术对于研究被认为具有类似功能的个体或蛋白质家族具有不可估量的价值。此外，它非常适合分子替换策略，该策略可以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精神、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。