Figure - available from: Scientific Reports
This content is subject to copyright. Terms and conditions apply.
![Observation of aggregated particles after LV pulse application and their role in the enhancement of GFP conversion in infected L929 cells. (a) Aggregated particle observation by optical microscopy (magnification: × 200), after application of twenty LV pulses on aliquots of three different solutions: (1) negative control (S-MEM unpulsed), (2) S-MEM pulsed, (3) PBS pulsed, (4) Hepes/NaCl pulsed. Size scale corresponding to the black bar is 80 µm. Photo editing was realized with Gimp 2.1 software. (b) Role of aggregated particles in the enhancement of GFP gene expression. L929 cells were infected with rAd (final MOI of 10⁴ vp/cell) using three types of inoculum: (1) viral suspension made in crude culture medium without any electric pulse (med + rAd); (2) viral suspension made using culture medium pulsed prior to virus mixing (med/LV + rAd); (3) viral suspension made in crude culture medium but pulsed after virus mixing ([med + rAd]/LV); for the last experimental condition, LV pulses were directly applied on cells, already mixed with viral suspension ([med + rAd + cells]/LV). In addition, for the first three experimental conditions, the viral suspension was either left 10 min at 4 °C (unfractionated inoculum indicated in grey) or subjected to a short centrifugation (300×g, 10 min, 4 °C) in Eppendorf tubes, yielding a upper (40 µl) and a lower (10 µl) fractions (indicated in white and black, respectively). Each fraction was then separately added to L929 target cells and GFP cell conversion was assessed by flow cytometry 24 h post treatment. UP unpulsed, LV application of low voltage pulses, med medium, rAd recombinant adenovirus. Data are presented as bar chart, with means ± SD (n = 3, except ultimate condition where n = 4). Statistical significances: *p < 0.05, **p < 0.01, and ***p < 0.001 stand for comparisons between experimental conditions (without fractionation) indicated by the brackets on the graph (one-way ANOVA with Bonferroni’s post-test). (c) The ratios of GFP cell conversion induced by the upper versus the lower fraction are presented for the first three experimental conditions as vertical boxes (minimum to maximum value), with central line indicating the mean and Y-axis indicating the ratio values. Statistical significances: *p < 0.05 and **p < 0.01 stand for the ratio comparisons indicated by the brackets on the graph (one-way ANOVA with Bonferroni's post-test).](publication/354384434/figure/fig4/AS:1065273717186596@1630992454961/Observation-of-aggregated-particles-after-LV-pulse-application-and-their-role-in-the.png)
Observation of aggregated particles after LV pulse application and their role in the enhancement of GFP conversion in infected L929 cells. (a) Aggregated particle observation by optical microscopy (magnification: × 200), after application of twenty LV pulses on aliquots of three different solutions: (1) negative control (S-MEM unpulsed), (2) S-MEM pulsed, (3) PBS pulsed, (4) Hepes/NaCl pulsed. Size scale corresponding to the black bar is 80 µm. Photo editing was realized with Gimp 2.1 software. (b) Role of aggregated particles in the enhancement of GFP gene expression. L929 cells were infected with rAd (final MOI of 10⁴ vp/cell) using three types of inoculum: (1) viral suspension made in crude culture medium without any electric pulse (med + rAd); (2) viral suspension made using culture medium pulsed prior to virus mixing (med/LV + rAd); (3) viral suspension made in crude culture medium but pulsed after virus mixing ([med + rAd]/LV); for the last experimental condition, LV pulses were directly applied on cells, already mixed with viral suspension ([med + rAd + cells]/LV). In addition, for the first three experimental conditions, the viral suspension was either left 10 min at 4 °C (unfractionated inoculum indicated in grey) or subjected to a short centrifugation (300×g, 10 min, 4 °C) in Eppendorf tubes, yielding a upper (40 µl) and a lower (10 µl) fractions (indicated in white and black, respectively). Each fraction was then separately added to L929 target cells and GFP cell conversion was assessed by flow cytometry 24 h post treatment. UP unpulsed, LV application of low voltage pulses, med medium, rAd recombinant adenovirus. Data are presented as bar chart, with means ± SD (n = 3, except ultimate condition where n = 4). Statistical significances: *p < 0.05, **p < 0.01, and ***p < 0.001 stand for comparisons between experimental conditions (without fractionation) indicated by the brackets on the graph (one-way ANOVA with Bonferroni’s post-test). (c) The ratios of GFP cell conversion induced by the upper versus the lower fraction are presented for the first three experimental conditions as vertical boxes (minimum to maximum value), with central line indicating the mean and Y-axis indicating the ratio values. Statistical significances: *p < 0.05 and **p < 0.01 stand for the ratio comparisons indicated by the brackets on the graph (one-way ANOVA with Bonferroni's post-test).
Source publication
Gene electrotransfer is an attractive method of non-viral gene delivery. However, the mechanism of DNA penetration across the plasma membrane is widely discussed. To explore this process for even larger structures, like viruses, we applied various combinations of short/long and high/low-amplitude electric pulses to L929 cells, mixed with a human ad...
Citations
... Pre-exposing the medium to EP (without exposing the cells or AAV to EP) yielded transduction outcomes that were identical to conventional experiments that exposed the cells only to AAV vectors without any EP. Ref. [26] reported enhanced gene transfer from pre-exposing cell culture medium to EP. In that case, the authors revealed the enhancement was due to impurity ions generated by an electrochemical reaction between the electrolytic medium and their aluminum electrodes. ...
We demonstrate that applying electric field pulses to hepatocytes, in vitro, in the presence of enhanced green fluorescent protein (EGFP)-expressing adeno-associated virus (AAV8) vectors reduces the viral dosage required for a given transduction level by more than 50-fold, compared to hepatocytes exposed to AAV8-EGFP vectors without electric field pulse exposure. We conducted 48 experimental observations across 8 exposure conditions in standard well plates. The electric pulse exposures involved single 80-ms pulses with 375 V/cm field intensity. Our study suggests that electric pulse exposure results in enhanced EGFP expression in cells, indicative of increased transduction efficiency. The enhanced transduction observed in our study, if translated successfully to an in vivo setting, would be a promising indication of potential reduction in the required dose of AAV vectors. Understanding the effects of electric field pulses on AAV transduction in vitro is an important preliminary step.
