Mol Ther Nucleic Acids. 2013 Mar 26;2(3):e82. doi: 10.1038/mtna.2013.10.
We present here a high efficiency, high viability siRNA-delivery method using a voltage-controlled chemical transfection strategy to achieve modulated delivery of polyethylenimine (PEI) complexed with siRNA in an in vitro culture of neuro2A cells and neurons. Low voltage pulses were applied to adherent cells before the administration of PEI-siRNA complexes. Live assays of neuro2a cells transfected with fluorescently tagged siRNA showed an increase in transfection efficiency from 62 ± 14% to 98 ± 3.8% (after -1 V). In primary hippocampal neurons, transfection efficiencies were increased from 30 ± 18% to 76 ± 18% (after -1 V). Negligible or low-level transfection was observed after preconditioning at higher voltages, suggesting an inverse relationship with applied voltage. Experiments with propidium iodide ruled out the role of electroporation in the transfection of siRNAs suggesting an alternate electro-endocytotic mechanism. In addition, image analysis of preconditioned and transfected cells demonstrates siRNA uptake and loading that is tuned to preconditioning voltage levels. There is approximately a fourfold increase in siRNA loading after preconditioning at -1 V compared with the same at ±2-3 V. Modulated gene expression is demonstrated in a functional knockdown of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in neuro2A cells using siRNA. Cell density and dendritic morphological changes are also demonstrated in modulated knockdown of brain derived neurotrophic factor (BDNF) in primary hippocampal neurons. The method reported here has potential applications in the development of high-throughput screening systems for large libraries of siRNA molecules involving difficult-to-transfect cells like neurons.Molecular Therapy-Nucleic Acids (2013) 2, e82; doi:10.1038/mtna.2013.10; published online 26 March 2013.
我们在此提出了一种高效率、高细胞存活率的 siRNA 传递方法，使用电压控制的化学转染策略，在体外培养的神经 2A 细胞和神经元中实现聚乙二烯亚胺（PEI）复合物与 siRNA 的调制传递。在给予 PEI-siRNA 复合物之前，向贴壁细胞施加低电压脉冲。用荧光标记的 siRNA 转染的神经 2a 细胞的活体测定显示，转染效率从 62±14%增加到 98±3.8%（-1V 后）。在原代海马神经元中，转染效率从 30±18%增加到 76±18%（-1V 后）。在施加更高电压的预处理后观察到可忽略或低水平的转染，表明与施加电压呈反比关系。用碘化丙啶进行的实验排除了 siRNA 转染中电穿孔的作用，表明存在替代的电内吞机制。此外，预处理和转染细胞的图像分析表明，siRNA 的摄取和加载可根据预处理电压水平进行调节。与相同的±2-3V 相比，在-1V 预处理后，siRNA 的加载量增加了约四倍。在神经 2A 细胞中，通过 siRNA 对甘油醛 3-磷酸脱氢酶（GAPDH）进行功能敲低，证明了基因表达的调节。在原代海马神经元中，对脑源性神经营养因子（BDNF）进行调制性敲低，也证明了细胞密度和树突形态的变化。本文报道的方法有可能应用于开发高通量筛选系统，用于涉及神经元等难以转染的细胞的大型 siRNA 分子文库。分子治疗-核酸（2013 年）2，e82；doi:10.1038/mtna.2013.10；在线发表于 2013 年 3 月 26 日。
