Developing RNA Interference for Gene Specific Silencing in Aplysia Neurons

开发用于海兔神经元基因特异性沉默的 RNA 干扰

基本信息

批准号：
7392756
负责人：
Kelsey C Martin
金额：
$ 20.63万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-04-01 至 2010-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7392756
关键词：
Afferent Neurons Alzheimer&apos s Disease Animals Aplysia Bathing Behavior Behavioral Behavioral Genetics Biological Biological Assay Biological Models Candidate Disease Gene Cells Chemicals Chromosome Pairing Clinical DNA Databases Disease Double-Stranded RNA Drug Addiction Electroporation Exploratory/Developmental Grant Expressed Sequence Tags Fluorescence Funding Ganglia Gene Expression Gene Silencing Gene Targeting Genes Genetic Screening Genome Goals Grant Green Fluorescent Proteins Guidelines In Situ Hybridization Individual Lead Learning Letters Mammals Mediating Memory Memory Loss Mental Retardation Mental disorders Methodology Methods Microinjections Molecular Motor Neurons Myxoid cyst Neurologic Neuronal Plasticity Neurons Numbers Polymerase Chain Reaction Preparation Process Protein Overexpression RNA RNA Interference Research Personnel Role Role playing therapy Sensory Small Interfering RNA Specificity Synapses Synaptic Transmission Synaptic plasticity System Techniques Technology Testing Transfection United States National Institutes of Health Work age related base expression vector genetic analysis genome sequencing immunocytochemistry improved innovation neuropsychiatry novel research study small hairpin RNA synaptogenesis therapeutic target

项目摘要

DESCRIPTION (provided by applicant): The goal of this R21 exploratory grant is to systematically develop the use of RNA interference (RNAi) in Aplysia neurons. Electrophysiological and behavioral studies in Aplysia have delineated the circuitry mediating simple forms of learning and memory in the animal. Cultured sensory-motor neurons from Aplysia have provided a model system for elucidating many of the molecular and cell biological mechanisms underlying learning-related synaptic plasticity. These mechanisms have been found to be generalizable to learning-related neuronal plasticity across species. While Aplysia offers many experimental advantages for cell biological and electrophysiological studies, it has not been suitable for genetic analyses. RNAi technology promises to transform Aplysia into a system in which genetic, behavioral, electrophysiological and cell biological analyses can be performed both in the animal and at the level of single cells and synapses. The experiments outlined in this proposal are aimed at developing methodologies for the use of RNAi in Aplysia. We will focus on investigating and optimizing 1) the type of RNA used for RNAi-long double stranded RNA (dsRNA) or small interfering RNAs (siRNAs)-- and 2) the method of delivery of the RNAi. To do this, we will target four endogenous Aplysia genes as well as exogenously overexpressed destabilized eGFP. We will determine whether the RNAi effectively silences target genes and whether or not this silencing is specific to the target gene. In addition, our experiments will identify the most efficient means of delivering RNA for RNAi and the best techniques for assaying the efficacy and specificity of RNAi-mediated gene silencing in Aplysia neurons. The results of the proposed experiments will be invaluable to researchers working in the Aplysia model system. From a broader perspective, the ability to use RNAi in Aplysia will generate valuable information about the molecular mechanisms underlying synapse formation, synaptic transmission and synaptic plasticity. This information likely will lead to the identification of potential therapeutic targets for the many neurological and psychiatric diseases in which these fundamental processes are perturbed. We propose to improve methods to study the molecular basis of learning and memory. The technologies we propose to develop will identify genes that are required for learning and in so doing will lead to potential therapies for the many diseases in which learning and memory are altered. Such diseases include mental retardation, age-related memory loss, Alzheimer's disease, drug addiction as well as many neuropsychiatric diseases.

描述（由申请人提供）：此R21探索性赠款的目标是系统地开发RNA干扰（RNAI）在Aplysia神经元中的使用。垂体中的电生理学和行为研究已经描绘了介导动物中简单的学习和记忆形式的电路。来自Aplysia的培养的感觉运动神经元提供了一个模型系统，用于阐明与学习相关的突触可塑性下的许多分子和细胞生物学机制。已经发现这些机制可以推广到各种物种之间与学习相关的神经元可塑性。虽然Aplysia为细胞生物学和电生理学研究提供了许多实验优势，但它并不适合遗传分析。 RNAi技术有望将垂直溶液转变为一个系统，在该系统中，可以在动物和单个细胞和突触的水平上进行遗传，行为，电生理和细胞生物学分析。该提案中概述的实验旨在开发用于在Aplysia中使用RNAi的方法。我们将专注于研究和优化1）用于RNAi长的双链RNA（DSRNA）或小干扰RNA（siRNA）的RNA类型 - 和2）RNAi的递送方法。为此，我们将瞄准四个内源性垂直基因以及外源过表达的不稳定的EGFP。我们将确定RNAi是否有效地沉默了靶基因以及该沉默是否针对靶基因。此外，我们的实验将确定为RNAi提供RNA的最有效方法，以及分析RNAi介导的基因沉默在Aplysia神经元中的功效和特异性的最佳技术。对Aplysia模型系统的研究人员来说，提出的实验的结果将是无价的。从更广泛的角度来看，在Aplysia中使用RNAi的能力将产生有关突触形成，突触传播和突触可塑性的分子机制的有价值信息。这些信息可能会导致对许多基本过程受到干扰的许多神经和精神病疾病的潜在治疗靶标。我们建议改善研究学习和记忆的分子基础的方法。我们建议开发的技术将识别学习所需的基因，因此，这样做将导致许多改变学习和记忆的疾病的潜在疗法。这些疾病包括智力降低，与年龄相关的记忆丧失，阿尔茨海默氏病，药物成瘾以及许多神经精神疾病。