Single-cell electroporation of adult sensory neurons for gene screening with RNA interference mechanism. - Archive ouverte HAL Access content directly
Journal Articles Journal of Neuroscience Methods Year : 2008

Single-cell electroporation of adult sensory neurons for gene screening with RNA interference mechanism.

Abstract

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.
Fichier principal
Vignette du fichier
boudes2008.pdf (304.74 Ko) Télécharger le fichier
Loading...

Dates and versions

inserm-00257563 , version 1 (19-02-2008)

Identifiers

Cite

Mathieu Boudes, Simon Pieraut, Jean Valmier, Patrick Carroll, Frédérique Scamps. Single-cell electroporation of adult sensory neurons for gene screening with RNA interference mechanism.. Journal of Neuroscience Methods, 2008, 170 (2), pp.204-11. ⟨10.1016/j.jneumeth.2008.01.018⟩. ⟨inserm-00257563⟩
78 View
449 Download

Altmetric

Share

Gmail Facebook Twitter LinkedIn More