Transfer of small interfering RNA by single-cell electroporation in cerebellar cell cultures
Transfer of small interfering RNA by single-cell electroporation in cerebellar cell cultures
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DOI：
10.1016/j.jneumeth.2008.11.025
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发表时间：
2009-03-30
影响因子：
3
通讯作者：
Hirashima, Naohide
中科院分区：
文献类型：
--
作者：
Tanaka, Masahiko;Yanagawa, Yuchio;Hirashima, Naohide
RNA干扰(RNAi)是研究神经元分化和变性相关基因功能的有力手段。与广泛使用的将小干扰RNA(SiRNA)导入细胞的方法不同，最近发展的单细胞电穿孔使siRNA能够转移到单个和已识别的细胞中。为了探索单细胞电穿孔siRNA的可行性，我们利用微管单细胞电穿孔技术将针对绿色荧光蛋白(GFP)的siRNA导入小脑细胞培养的GFP表达的高尔基细胞和浦肯野细胞中。在相同的电穿孔细胞中，实时监测GFP荧光强度的时间变化，直到电穿孔后4d。为了最大化RNAi的效率和电穿孔细胞的活力，优化了几个参数，包括微管的尖端直径和电阻，siRNA和荧光染料标记的浓度，脉冲的电压和时间。在最佳条件下，GFP siRNA的转移显著降低了电穿孔细胞中的GFP荧光，而阴性对照siRNA对GFP荧光没有影响。GFP siRNA在浦肯野细胞中比在高尔基细胞中更有效。电穿孔的浦肯野细胞形态正常，包括细小的树突。因此，单细胞电穿孔siRNA可能是一种简单而有效的工具，在神经元原代培养中沉默单个细胞的基因表达。此外，这种方法引入的siRNA的基因沉默和非靶向效应可能因神经细胞类型而异，单细胞电穿孔的参数应在每种细胞类型中进行优化。(C)2008爱思唯尔B.V.保留所有权利。
RNA interference (RNAi) is a powerful means to investigate functions of genes involved in neuronal differentiation and degeneration. In contrast to widely used methods for introducing small interfering RNA (siRNA) into cells, recently developed single-cell electroporation has enabled transfer of siRNA into single and identified cells. To explore the availability of single-cell electroporation of siRNA in detail, we introduced siRNA against green fluorescent protein (GFP) into GFP-expressing Golgi and Purkinje cells in cerebellar cell cultures by single-cell electroporation using micropipettes. The temporal changes in the intensity of GFP fluorescence in the same electroporated cells were monitored in real-time up to 4 days after electroporation. Several parameters, including tip diameter and resistance of micropipettes, concentrations of siRNA and a fluorescent dye marker, voltage and time of pulses, were optimized to maximize both the efficacy of RNAi and the viability of the electroporated cells. Under the optimal conditions, transfer of GFP siRNA significantly reduced GFP fluorescence in the electroporated cells, whereas that of negative control siRNA had no effects. GFP siRNA was more efficient in Purkinje cells than in Golgi cells. The electroporated Purkinje cells were normal in their morphology, including elaborated dendrites. Thus, the single-cell electroporation of siRNA could be a simple but effective tool for silencing gene expression in individual cells in neuronal primary cultures. In addition, both gene-silencing and off-target effects of siRNA introduced by this method may differ between neuronal cell types, and the parameters of single-cell electroporation should be optimized in each cell type. (C) 2008 Elsevier B.V. All rights reserved.