Transfer of small interfering RNA by single-cell electroporation in cerebellar cell cultures
ABSTRACT 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.
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ABSTRACT: Single-cell electroporation is a technique for transfecting individual cells in tissue culture at relatively high efficiencies, however it is both time-consuming and low-throughput and this limits the number of different labeling agents that can be effectively introduced into a region of tissue in reasonable periods of time. A novel system that will rapidly load, clean, and accurately position a glass micropipette electrode into tissue culture for single-cell electroporation is proposed. The system will significantly increase the number of different labeling agents that can be introduced into a single tissue culture per unit time. This in turn, will provide a means for improving the study of neural anatomy at cellular resolutions in both tissue culture and in vivo environments.05/2010;
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ABSTRACT: The development and application of single neuron electroporation largely advanced the use of traditional genetics in investigations of the central nervous system. This quick and accurate manipulation of the brain at individual neuron level allowed the gain and loss of functional analyses of different genes and/or proteins. This manuscript reviewed the development of the technique and discussed some technical aspects in practical manipulations. Then the manuscript summarized the potential applications with this technique. Last but not least, the technique showed prospective future when combined with other modern methods in neuroscience research.International Archives of Medicine 11/2010; 3:28.