Transfer of small interfering RNA by single-cell electroporation in cerebellar cell cultures.
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.
Tanaka
Masahiko
M
Department of Cellular Biophysics, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan. mtanaka@phar.nagoy-cu.ac.jp
Yanagawa
Yuchio
Y
Hirashima
Naohide
N
D000818:Animals; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology*; D018274:Electroporation / Q000379:methods*; D020868:Gene Silencing; D018014:Gene Transfer Techniques; D005968:Glutamate Decarboxylase / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009474:Neurons / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors
D053496:Inositol 1,4,5-Trisphosphate Receptors; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins; D005968:Glutamate Decarboxylase; C402565:glutamate decarboxylase 1