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

描述（由申请人提供）：一种相对较新的遗传修饰生物体的方法涉及使用重组病毒感染体内离散细胞群，通过过表达目的转基因改变基因表达。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）来沉默目标群体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，因为基因操纵可以在感兴趣的特定大脑区域急剧引入，并且这些操纵可以在学习之前或之后创建。然而，为了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体中敲除超过50%的基因表达，这不是一个微不足道的壮举，因为靶细胞需要各自被跨越几毫米的大鼠大脑区域的多种病毒持续感染，以有效地敲除靶基因。因此，本研究的目的是改进这种方法，以促进杏仁核依赖性学习和记忆基因的研究。在我们的第一个目标中，我们将确定腺相关病毒（AAV），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确定哪些AAV血清型感染这些核内的兴奋性谷氨酸能、表达aCaMK II的神经元和表达抑制性gabaergic GAD 65的神经元，所述定制的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。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中包含的shRNA转基因的数量来增强病毒介导的体内基因敲减，以及这种修饰是否会导致学习和记忆的更大损害。具体地，AAV将被设计成具有不同数量的shRNA表达盒，所述shRNA表达盒被设计成靶向活性调节的细胞骨架蛋白（Arc）。此外，将设计一组对照病毒，以携带类似数量的不靶向任何转录物进行敲低的shRNA表达盒。单独的大鼠组将接受这些病毒的双侧输注 进入BLA。将大鼠进行恐惧条件化，并在训练后3小时和24小时测试听觉恐惧记忆的保持，以分别评估Arc敲除对短期和长期恐惧记忆保持的影响。将进行额外的实验以测量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将因Arc敲除而受损，并且LTM的受损将与Arc敲除的程度相关，因此与靶向Arc的病毒中存在的shRNA转基因的数量相关。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.