Abstract
BackgroundGene transfer into many cell types has been successfully used to develop alternative and adjunct approaches to conventional medical treatment. However, effective transfection of postmitotic neurons remains a challenge. The aim of this study was to develop a method for gene transfer into rat primary dorsal root ganglion neurons using sonoporation.MethodsDissociated cells from adult rat dorsal root ganglion (DRG) cells were sonicated for 1-8 s at 2.5-10 W to determine the optimal ultrasound duration and power for gene transfection and cell survival. Transfection efficiency was compared between sonoporation, liposome and lentiviral vector gene transfer techniques.ResultsThe optimum ultrasound intensity was 5 W for 2 s and yielded an efficiency of gene transfection of 31% and a survival rate of 35%.ConclusionsSonoporation can be optimized to minimize cell death and yield a high percentage of transfected neurons and that this technique can be easily applied to primary cultures of rat dorsal root ganglion neurons.
Highlights
Gene transfer into many cell types has been successfully used to develop alternative and adjunct approaches to conventional medical treatment
Dissociated cells from adult rat dorsal root ganglion (DRG) were suspended at a density of 10,000 cells per cm3
At an energy output of 5 W and sonoporation for 1 s, 2 s, 4 s and 8 s, cells immunoreactive for β-III tubulin constituted 76%, 75%, 85% and 78%, respectively, of GFP-transfected cells (Figure 4). These findings show that sonoporation-mediated gene transfer is effective in DRG cells
Summary
Gene transfer into many cell types has been successfully used to develop alternative and adjunct approaches to conventional medical treatment. The aim of this study was to develop a method for gene transfer into rat primary dorsal root ganglion neurons using sonoporation. Many gene transfer methods have been developed in the past two decades, including calcium phosphate coprecipitation, microinjection, recombinant viruses, liposomemediated gene transfer, lipids that do not form liposomes, high molecular weight cationic polymers, particle bombardment (biollistics) and electroporation. Ultrasound can be used to modify the permeability of the cell membrane to facilitate the uptake of RNA [1,2,3,4,5] and DNA into the cell [6,7,8,9,10,11]. Lowfrequency ultrasound increases membrane permeability to many drugs, including high molecular weight proteins [12].
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