Grames Mychal S, Jackson Kasey L, Dayton Robert D, Stanford John A, Klein Ronald L
Department of Pharmacology, Toxicology, and Neuroscience, Louisiana State University Health Sciences Center.
Department of Molecular & Integrative Physiology, University of Kansas Medical Center.
J Vis Exp. 2017 Aug 25(126):55994. doi: 10.3791/55994.
Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), optogenetics, cre-lox targeting, etc. The purpose of this manuscript is to aid the investigator attempting expansive central nervous system (CNS) gene transfer in the rat via tail vein injection of AAV. Wide-scale expression is relevant for conditions with widespread pathology, and a rat model is significant due to its greater size and physiologic similarities to humans compared to mice. In this example application, a wide-scale neuronal transduction is used to mimic a neurodegenerative disease that affects the entire spinal cord, amyotrophic lateral sclerosis (ALS). The efficient wide-scale CNS transduction can also be used to deliver therapeutic protein factors in pre-clinical studies. After a post-injection expression interval of several weeks, the effects of the transduction are evaluated. For a green fluorescent protein (GFP) control vector, the amount of GFP in the cerebellum is estimated quickly and reliably by a basic imaging program. For motor disease phenotypes that are induced by the ALS related protein transactive response DNA-binding protein of 43 kDa (TDP-43), the deficits are scored by escape reflex and rotarod. Beyond disease modeling and gene therapy, there are diverse potential applications for the wide-scale gene targeting described here. The expanded use of this method will aid in expediting hypothesis testing in the neurosciences and neurogenetics.
腺相关病毒（AAV）载体是神经科学领域用于成簇规律间隔短回文重复序列（CRISPR）、光遗传学、cre-lox靶向等研究的关键试剂。本手稿旨在帮助研究人员通过尾静脉注射AAV在大鼠中进行广泛的中枢神经系统（CNS）基因转移。广泛表达适用于具有广泛病理学特征的疾病，大鼠模型因其体型较大且与人类在生理上更相似，相较于小鼠具有重要意义。在这个示例应用中，广泛的神经元转导被用于模拟一种影响整个脊髓的神经退行性疾病——肌萎缩侧索硬化症（ALS）。高效的广泛中枢神经系统转导还可用于在临床前研究中递送治疗性蛋白质因子。在注射后数周的表达期过后，评估转导的效果。对于绿色荧光蛋白（GFP）对照载体，通过一个基本的成像程序可快速可靠地估计小脑中GFP的含量。对于由43 kDa的ALS相关蛋白反式激活反应DNA结合蛋白（TDP-43）诱导的运动疾病表型，通过逃避反射和转棒试验对缺陷进行评分。除了疾病建模和基因治疗外，这里描述的广泛基因靶向还有多种潜在应用。这种方法的广泛应用将有助于加快神经科学和神经遗传学领域的假设检验。
