Single-cell electroporation of adult sensory neurons for gene screening with RNA interference mechanism.
RNA interference appears as a technique of choice to identify gene candidate or to evaluate gene function. To date, a main problem is to achieve high transfection efficiencies on native cells such as adult neurons. In addition, transfection on organ or mass culture does not allow to approach the cellular diversity. Dorsal root ganglia are composed with several cell types to convey somato-sensory sensations. Single-cell electroporation is the most recent method of transfection that allows the introduction into cells, not only dyes or drugs, but also large molecules such plasmid DNA expression constructs. In the present study, the application of the RNA interference technique with the use of single-cell electroporation was evaluated in primary culture of adult sensory neurons. With the use of fluorescent dextran as a co-transfectant, we first determined the non-specific siRNA concentration leading to cell death. Efficacy of siRNA at the non-toxic concentration was demonstrated at the protein level by extinction of GFP fluorescence in actin-GFP neurons and by the inhibition of the intracellular Cl- concentration increase following activation of the membrane co-transporter Na+-K+-2Cl- in regenerating axotomized sensory neurons. Altogether, these data show that delivery of siRNAs by single-cell electroporation leads to the induction of functional RNA interference.
Boudes
Mathieu
M
Inserm U583, Univ Montpellier II, Montpellier F-34000 France.
Pieraut
Simon
S
Valmier
Jean
J
Carroll
Patrick
P
Scamps
Frédérique
F
D000818:Animals; D002470:Cell Survival; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism; D004594:Electrophysiology; D018274:Electroporation / Q000379:methods*; D005260:Female; D005727:Ganglia, Spinal / Q000166:cytology; D049452:Green Fluorescent Proteins / Q000235:genetics; D007091:Image Processing, Computer-Assisted; D051379:Mice; D009475:Neurons, Afferent / Q000502:physiology*; D034741:RNA, Small Interfering; D014162:Transfection
D002712:Chlorides; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins