Long-Term Gene-Silencing Effects of siRNA Introduced by Single-Cell Electroporation into Postmitotic CNS Neurons
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To explore how long the gene-silencing effects of siRNA introduced into postmitotic neurons continue, we transferred siRNA against GFP into GFP-expressing Purkinje and Golgi cells in cerebellar cell cultures by single-cell electroporation. The temporal changes in the intensity of GFP fluorescence in the same electroporated cells were monitored in real time using GFP imaging. Under standard conditions, GFP fluorescence was reduced to under one-tenth of the initial levels 4–7 days after electroporation. Such effects continued at least up to 14 days after electroporation. The effects of siRNAs against endogenous genes also continued for the same period. Thus, this method could be an effective tool for silencing gene expression for a long period in postmitotic neurons.
KeywordssiRNA Postmitotic neuron Single-cell electroporation Cerebellum Purkinje cell Golgi cell
We thank Dr. N. Maeda for his kind gift of the anti-IP3R antibody. This study was supported in part by grants-in-aid from the MEXT of Japan, the Takeda Science Foundation and the Brain Science Foundation.