Optimization of Adeno-Associated Virus for the Study of Amygdala Dependent Learni

用于杏仁核依赖性学习研究的腺相关病毒的优化

基本信息

批准号：
8701412
负责人：
JONATHAN E PLOSKI
金额：
$ 7.65万
依托单位：
UNIVERSITY OF TEXAS DALLAS
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-01 至 2016-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8701412
关键词：
Affinity Amygdaloid structure Auditory Basal Cell Behavioral Bilateral Brain Brain region CMV promoter Cell Nucleus Cells Central Lateral Nucleus Custom Cytoskeletal Proteins Dependovirus Diffusion Effectiveness Fluorescence Microscopy Fright Funding Funding Mechanisms Gene Expression Gene Silencing Gene Targeting Genes Genetic Transcription Genetically Modified Organisms Glutamates Hour Impairment Infection Infusion procedures Instinct Lead Learning Measures Mediating Memory Memory impairment Mental disorders Methods Modeling Modification Molecular Molecular Biology Mus Nature Neurons Population Positioning Attribute Proteins RNA Interference Rattus Research Research Project Grants Risk Rodent Serotyping Synaptic plasticity System Target Populations Technology Testing Training Transcript Transgenes United States National Institutes of Health Viral Viral Vector Virus Western Blotting calmodulin-dependent protein kinase II cell type cohort conditioned fear design excitatory neuron experience genetic manipulation improved in vivo inhibitory neuron interest millimeter novel strategies overexpression promoter public health relevance recombinant virus red fluorescent protein research study small hairpin RNA tool

项目摘要

DESCRIPTION (provided by applicant): A relatively new approach to genetically modify organisms involves the use of recombinant viruses to infect discrete populations of cells in vivo to alter gene expression by overexpressing a transgene of interest. If the virus is designed to express a short hairpin RNA (shRNA), RNA interference technology (RNAi) can be utilized to silence specific genes of interest in the targeted population. This technology is especially useful for learning and memory research, since the genetic manipulations can be introduced acutely in specific brain regions of interest and these manipulations can be created before or after learning. However, for viral mediated gene silencing to truly be effective it must be able to knockdown gene expression more than 50% across a population of pertinent target cells in vivo, which is not a trivial feat since targeted cells need to each be infected by multiple viruses consistently across regions of the rat brain that can span millimeters to effectively knockdown the target gene. Therefore this proposal is designed to improve upon this approach to facilitate the study of genes in amygdala-dependent learning and memory. In our first aim we will identify the Adeno-associated virus (AAV), viral serotypes that maximally infect the rat Basal Lateral and Central nuclei of the Amygdala (BLA and CeA). Additionally we will determine which AAV serotypes infect excitatory glutamatergic, aCaMKII expressing neurons and inhibitory gabaergic GAD65 expressing neurons within these nuclei using custom-made viruses that will tag these differing neuronal types, allowing them to be distinguished from other cell types within the amygdala. In our second aim we will examine if viral-mediated in vivo gene knockdown can be enhanced by increasing the number of shRNA transgenes included within the virus and if this modification leads to a greater impairment in learning and memory. Specifically AAV will be designed to harbor varying numbers of shRNA expression cassettes designed to target the Activity Regulated Cytoskeletal protein (Arc). Additionally, a set of control viruses will be designed to harbor a similar number of shRNA expression cassettes that do not target any transcript for knockdown. Separate groups of rats will receive bilateral infusions of these viruses into the BLA. The rats will be fear conditioned and tested for retention of auditory fear memory at both 3 hours and 24 hours after training to evaluate the effect of Arc knockdown on the retention of short-term and long-term fear memory, respectively. Additional experiments will be conducted to measure locomotor behavior, innate fear, expression of fear and Arc protein levels within the BLA. We hypothesize that LTM will be impaired by Arc knockdown and the impairment in LTM will correlate with the degree of Arc knockdown and therefore the number of shRNA transgenes that were present in the virus targeting Arc. This RO3 research project will allow us to improve upon technology that will facilitate learning and memory research.

描述（由申请人提供）：一种相对较新的基因改造生物体的方法涉及使用重组病毒在体内感染离散的细胞群，通过过表达感兴趣的转基因来改变基因表达。如果病毒被设计为表达短发夹 RNA (shRNA)，则可以利用 RNA 干扰技术 (RNAi) 来沉默目标人群中感兴趣的特定基因。这项技术特别有用对于学习和记忆研究，因为基因操作可以被敏锐地引入到感兴趣的特定大脑区域，并且这些操作可以在学习之前或之后创建。然而，要使病毒介导的基因沉默真正有效，它必须能够在体内相关靶细胞群中敲低 50% 以上的基因表达，这并不是一件容易的事，因为靶细胞需要分别被多种病毒感染一致地跨过大鼠大脑的数毫米区域，以有效地敲除目标基因。因此，该提案旨在改进这种方法，以促进杏仁核依赖性学习和记忆基因的研究。我们的第一个目标是鉴定腺相关病毒 (AAV)，这种病毒血清型能够最大限度地感染大鼠杏仁核的基底外侧核和中央核（BLA 和 CeA）。此外，我们将使用定制病毒来确定哪些 AAV 血清型感染这些细胞核内的兴奋性谷氨酸、aCaMKII 表达神经元和抑制性 gabaergic GAD65 表达神经元，这些病毒将标记这些不同的神经元类型，从而将它们与杏仁核内的其他细胞类型区分开来。在我们的第二个目标中，我们将研究是否可以通过增加病毒内包含的 shRNA 转基因数量来增强病毒介导的体内基因敲除，以及这种修饰是否会导致学习和记忆能力受到更大的损害。具体来说，AAV 将被设计为包含不同数量的 shRNA 表达盒，这些表达盒旨在靶向活性调节细胞骨架蛋白 (Arc)。此外，一组对照病毒将被设计为含有相似数量的 shRNA 表达盒，这些表达盒不针对任何转录物进行敲低。不同组的大鼠将接受这些病毒的双侧输注进入 BLA。训练后3小时和24小时对大鼠进行恐惧条件反射并测试听觉恐惧记忆的保留，以分别评估Arc敲低对短期和长期恐惧记忆保留的影响。将进行额外的实验来测量 BLA 内的运动行为、先天恐惧、恐惧表达和 Arc 蛋白水平。我们假设 LTM 将受到 Arc 敲低的损害，并且 LTM 的损害将与 Arc 敲低的程度相关，从而与针对 Arc 的病毒中存在的 shRNA 转基因的数量相关。 RO3 研究项目将使我们能够改进促进学习和记忆研究的技术。