Voltage Preconditioning Allows Modulated Gene Expression in Neurons Using PEI-complexed siRNA.
Voltage Preconditioning Allows Modulated Gene Expression in Neurons Using PEI-complexed siRNA.
复制标题
电压预处理允许使用 PEI 复合 siRNA 调节神经元中的基因表达。
DOI：
10.1038/mtna.2013.10
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发表时间：
2013
期刊：
影响因子：
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通讯作者：
Muthuswamy,Jit
中科院分区：
文献类型：
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作者：
Sridharan,Arati;Patel,Chetan;Muthuswamy,Jit
我们提出了一种高效、高活性的siRNA递送方法，该方法采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了与siRNA复合的聚乙烯亚胺(PEI)的可调递送。在给药PEI-siRNA复合体之前，对贴壁细胞施加低电压脉冲。经−-1- -V处理后，神经2a细胞的转染率从62±14%提高到98±3.8%。原代海马神经元的转染率从30±18%提高到76±18%(−1 V后)。在较高电压下预适应后，观察到可忽略或低水平的转染率，这表明与施加的电压成反比。使用碘化丙啶的实验排除了电穿孔在siRNAs转移中的作用，这表明了另一种电子内吞机制。此外，对预适应和转基因细胞的图像分析表明，siRNA的摄取和负载与预适应电压水平有关。在−1 V时，与在±2-3 V时相比，预适应后的小干扰RNA负载量大约增加了4倍。在原代海马神经元中，脑源性神经营养因子(BDNF)的调节敲除也显示出细胞密度和树突形态的变化。本文报道的方法在高通量筛选大容量siRNA分子文库方面具有潜在的应用前景，这些文库涉及像神经元这样的难以转化的细胞。
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 anin vitroculture 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.