Sonoporation-mediated gene transfer into adult rat dorsal root ganglion cells

Abstract

Background: Gene 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.

Methods: Dissociated 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.

Results: The optimum ultrasound intensity was 5 W for 2 s and yielded an efficiency of gene transfection of 31% and a survival rate of 35%.

Conclusions: Sonoporation 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.

Figure 1

Optimal conditions for DRG transfection via sonoporation. Dissociated cells from adult rat DRGs were suspended at a density of 10,000 cells per cm³ in a solution containing 10 μg/ml of pE-GFP C1 and were sonicated for 1 s at 2.5-10 W using a 12 mm diameter probe tip. The cells were maintained in vitro for 48 h after sonoporation. The calibration bar in Panel c represents 100 μm and applies to Panels a-c. Panel d shows the number of GFP-expressing cells per 5 mm² after sonication at various energy levels. Panel e shows the number of GFP-expressing cells per 5 mm² after sonication for various sonication durations at 5 W.

Figure 2

Cell survival for various sonoporation durations and energy levels. Survival was defined as the number of calcein-positive cells 2 d after sonoporation and expressed as a percentage of the number of calcein-positive cells in the unsonoporated control. Results are expressed as the mean ± SD. *p < 0.05.

Figure 3

Comparison of gene transfection efficiency between the sonoporation and lentiviral vector methods. The histogram shows the relative number of transfected GFP-positive cells per 5 mm² after sonoporation (left side of the panel) and gene transfer using the lentiviral vector (right side of the panel). All DRG cells were derived from the same pool of cells.

Figure 4

β-tubulin III- and GFP-immunoreactive DRG cells. After addition of 10 μg/ml of pE-GFP C1, the cells were sonicated for 2 s at 5 W using a 12 mm probe. The cells were processed for immunocytochemistry 48 h after sonoporation and labeled with antibodies against GFP and β-tubulin III (a marker of neuronal cells).