Abstract
Short interfering RNAs (siRNAs) represent new potential therapeutic tools owing to their capacity to induce strong, sequence-specific, gene silencing in cells. Electropulsation is one of the physical methods successfully used to transfer siRNA into living cells in vitro and in vivo. Although this approach is proved to be effective for silencing gene expression by RNA interference, very little is known about the basic processes supporting siRNA transfer. In this study, we investigated, by direct visualization at the single-cell level, the electro-delivery of Alexa Fluor 546-labeled siRNA into murine melanoma cells stably expressing the enhanced green fluorescent protein (EGFP) as a target gene. The electrotransfer of siRNA was quantified by time-lapse fluorescence microscopy and was correlated with the silencing of EGFP expression. A direct transfer into the cell cytoplasm of the negatively charged siRNA was observed across the plasma membrane exclusively on the side facing the cathode. The oligonucleotide then freely diffused across the cytosol. Therefore, we show that the electric field pulse acts on both the permeabilization of the cell plasma membrane and on the electrophoretic drag of the negatively charged siRNA molecules from the bulk phase into the cytoplasm. The mechanism involved was clearly specific for the physicochemical properties of the electrotransferred molecule and was different from that observed with the electro-transfer of small molecules or plasmid DNA.
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Acknowledgments
The authors would like to thank laboratory members for their help and comments (Elisabeth Bellard, Aurelie Paganin-Gioanni, Sophie Chabot, Sandrine Pelofy, and Marie-Pierre Rols). Financial supports were obtained from the Seventh Framework European Programme (FP7) OncomiR [grant number 201102], the “Ligue nationale contre le Cancer,” and the “Région Midi-Pyrenées.” This work has been performed in collaboration with the “Toulouse Réseau Imagerie” core IPBS facility (Genotoul, Toulouse, France), which is supported by the Association Recherche Cancer (n°5585), Region Midi Pyrenees (CPER), and Grand Toulouse cluster. This work was conducted in the scope of the LEA-EBAM and the EU-COST Action TD1104.
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Teissie, J., Golzio, M. (2014). Direct Imaging of siRNA Electrotransfer at the Single-Cell Level. In: Li, S., Cutrera, J., Heller, R., Teissie, J. (eds) Electroporation Protocols. Methods in Molecular Biology, vol 1121. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4614-9632-8_10
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DOI: https://doi.org/10.1007/978-1-4614-9632-8_10
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