Alzate-Correa Diego, Lawrence William, Higuita-Castro Natalia, Gallego-Perez Daniel
Department of Biomedical Engineering, The Ohio State University, Columbus, OH, USA.
Biomedical Science Graduate Program, The Ohio State University, Columbus, OH, USA.
Methods Mol Biol. 2020;2050:145-152. doi: 10.1007/978-1-4939-9740-4_15.
Developing effective gene therapies for disorders of the central nervous system (CNS) is extremely challenging due to the lack of safe and efficient gene delivery methods to neurons and glial cells, hampering the study of CNS physiology and the identification of novel therapeutic targets. Current gene transfer methodologies for neuronal cultures rely on synthetic nanoparticles or viral transduction. These approaches present low gene transfer efficiency, are highly toxic, and may induce adverse immune responses. Electroporation has been implemented as an alternative approach; however, this method is restricted for the most part to cells in suspension, and electrical overstimulation of the neuronal membrane may have detrimental consequences. To overcome these barriers, here we describe the implementation of nanochannel-based electroporation for gene delivery into primary neural cultures safely and efficiently. We outline the preparation of viable primary neuronal cultures from the hippocampus of E18.5 mouse embryos and describe the optimal parameter for transfection using a nanochannel-based electroporation platform.
由于缺乏向神经元和神经胶质细胞进行安全有效基因递送的方法，开发针对中枢神经系统（CNS）疾病的有效基因疗法极具挑战性，这阻碍了中枢神经系统生理学研究和新型治疗靶点的识别。目前用于神经元培养的基因转移方法依赖于合成纳米颗粒或病毒转导。这些方法基因转移效率低、毒性高，且可能引发不良免疫反应。电穿孔已作为一种替代方法实施；然而，该方法在很大程度上仅限于悬浮细胞，并且对神经元膜的过度电刺激可能会产生有害后果。为了克服这些障碍，我们在此描述基于纳米通道的电穿孔技术，用于安全有效地将基因递送至原代神经培养物中。我们概述了从E18.5小鼠胚胎海马制备有活力的原代神经元培养物的方法，并描述了使用基于纳米通道的电穿孔平台进行转染的最佳参数。
