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

描述(申请人提供)：一种相对较新的对生物进行基因改造的方法涉及使用重组病毒感染体内离散的细胞群体，通过过表达目的基因来改变基因表达。如果病毒被设计成表达短发夹状RNA(ShRNA)，RNA干扰技术(RNAi)可以用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特定的大脑感兴趣区域，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的靶细胞群体中击倒50%以上的基因表达，这不是一项微不足道的壮举，因为靶细胞需要每个靶细胞都一致地感染多个病毒，跨越大鼠大脑的各个区域，这些区域可以跨越几毫米长，以有效地击倒靶基因。因此，这项建议旨在改进这一方法，以促进杏仁核依赖学习和记忆基因的研究。在我们的第一个目标中，我们将确定腺相关病毒(AAV)，这是一种病毒血清型，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，我们将使用定制的病毒来确定哪些AAV血清型会感染这些核团中兴奋性谷氨酸能神经元、aCaMKII表达神经元和抑制性GABA能GAD65表达神经元，这些病毒将标记这些不同的神经元类型，使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，我们将检查是否可以通过增加病毒中包含的shRNA转基因的数量来增强病毒介导的体内基因敲除，以及这种修改是否会导致学习和记忆的更大损害。具体地说，AAV将被设计成携带不同数量的shRNA表达盒，旨在靶向活性调节的细胞骨架蛋白(Arc)。此外，一组对照病毒将被设计为携带类似数量的shRNA表达盒，这些表达盒不针对任何转录本进行敲除。不同组的大鼠将接受双侧注射这些病毒 进入了血防中心。在训练后3小时和24小时，对大鼠进行恐惧条件化和听觉恐惧记忆保持测试，以分别评估Arc基因敲除对短期和长期恐惧记忆保持的影响。还将进行其他实验，以测量运动行为、先天恐惧、恐惧表达和BLA内的Arc蛋白水平。我们假设LTM将受到Arc基因敲除的损害，LTM的损害将与Arc敲除的程度相关，从而与靶向Arc的病毒中存在的shRNA转基因数量相关。这个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。

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.