Developing RNA Interference for Gene Specific Silencing in Aplysia Neurons

描述（由申请人提供）：这项R21探索性资助的目标是系统地开发RNA干扰（RNAi）在海兔神经元中的应用。澳大利亚的电生理学和行为学研究已经描绘出了动物体内调节简单形式的学习和记忆的电路。培养的猕猴桃感觉运动神经元为阐明学习相关突触可塑性的分子和细胞生物学机制提供了一个模型系统。这些机制已经被发现可以推广到跨物种学习相关的神经元可塑性。虽然应用于细胞生物学和电生理学研究中具有许多实验优势，但它不适合用于遗传分析。RNAi技术有望将applysia转变为一个系统，在该系统中，可以在动物和单细胞和突触水平上进行遗传、行为、电生理和细胞生物学分析。本提案中概述的实验旨在开发在澳大利亚使用RNAi的方法。我们将重点研究和优化1)用于RNAi的RNA类型-长双链RNA （dsRNA）或小干扰RNA (sirna)-以及2)RNAi的递送方法。为了做到这一点，我们将针对四种内源的泛蓝基因以及外源过表达的不稳定的eGFP。我们将确定RNAi是否有效地沉默靶基因，以及这种沉默是否针对靶基因。此外，我们的实验将确定为RNAi传递RNA的最有效手段，以及分析RNAi介导的基因沉默在海兔神经元中的有效性和特异性的最佳技术。提出的实验结果将是宝贵的研究人员工作在Aplysia模型系统。从更广泛的角度来看，在applysia中使用RNAi的能力将为突触形成、突触传递和突触可塑性的分子机制提供有价值的信息。这一信息可能会导致确定许多神经和精神疾病的潜在治疗靶点，其中这些基本过程受到干扰。我们建议改进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，这样做将为许多学习和记忆改变疾病带来潜在的治疗方法。这些疾病包括智力迟钝、与年龄有关的记忆丧失、阿尔茨海默病、吸毒成瘾以及许多神经精神疾病。

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.