# Efficient Gene Transfection by Electroporation--In Vitro and In Silico Study of Pulse Parameters
## 4 Discussion
### Time Dynamics of pDNA Expression  
Measurements of pDNA expression every 8 h over a six-day period (Figures 10 and 11) show that the onsets of GFP expression (both percentage of GFP-positive cells and their MFI) are comparable for the 200 ns, HF-BP, 8 \(\times\) 100 \(\upmu\)s, and 8 \(\times\) 5 ms pulse protocols but depend greatly on the cell line. Similar time dynamics of the GET and MFI for all pulse durations in one cell line suggest that all pulse protocols triggered the same mechanisms responsible for pDNA translocation through the cytoplasm to the cell nucleus. In C2C12 myoblasts, the GFP-positive cells and their MFI reached a peak soon after GET and then declined steadily, falling below 10% for all four pulse durations at day 6. Similar time dynamics were observed for human mesenchymal stem cells [86]. On the contrary, in 1306 fibroblasts, peaks of the maximum GFP-positive cells and their MFI were broad and not very pronounced. After reaching a peak, the percentage of GFP-positive cells started to decrease slowly. On day 6 after GET with all pulse durations, around half of the cells were still GFP-positive (Figures 10 and 11). Differences in time dynamics and duration of transgene expression after GET between cell lines have been reported previously [99]. Differences in curves for the GET and MFI for all pulse protocols between cell lines suggest that mechanisms of pDNA translocation through the cytoplasm to the cell nucleus and the duration of the transcription of pDNA are most probably different in different cell lines. The lower percentage of GFP-positive cells and their lower MFIs in C2C12 myoblasts could be a consequence of the higher number or degree of activation of DNA sensors in this cell line, which could trigger pDNA degradation to a larger extent compared to 1306 fibroblasts. During GET, pDNA could activate endosomal DNA sensors during translocation to the nucleus with endocytosis mediated pathways or cytoplasmic DNA sensors by entering the cell through cell membrane defects caused during pulse delivery. Additionally, cytosolic DNA sensors might be triggered by pDNA released to cytosol after endosomal escape. It was already shown that GET with pDNA led to the upregulation of several proposed cytosolic DNA sensors in different tumor cell types [78; 100] and also in C2C12 myoblasts [82]. Other reasons for the observed decrease in transgene expression over time in C2C12 myoblasts could be the loss of the pDNA through the nuclear pores, loss of the pDNA at each mitosis, de novo pDNA methylation preventing pDNA transcription, or pDNA degradation by endonucleases [80]. We continuously observed differences in the overall GET and MFI between the two cell lines studied. These differences might be the consequence of variations in cell membrane composition [101], endocytic pathways specific to certain cell lines, or the degree of activation of endocytic pathways [102; 103], as well as the presence of cytosolic nucleases [104]. Our results show that MFI of GFP-positive cells is more unpredictable and unrepeatable than the percentage of GFP-positive cells. The MFI of 1306 fibroblasts in our experiments with increasing pDNA concentration (Figure 3D) was much lower than the MFI of 1306 fibroblasts transfected with the same concentration (500 \(\upmu\)g/mL) of pDNA and with the same pulse protocols in the experiments of time dynamics of pDNA expression (Figure 11B). At the same time, the GET efficiency in both experiments was comparable with the same pulse protocols. Contrarily, in C2C12 myoblasts, the MFI was comparable in both experiments, but GET after 8 \(\times\) 5 ms was much lower in the experiments of the time dynamics of the pDNA expression (Figure 10A) compared to the experiments with increasing pDNA concentration (Figure 3A). It was shown previously that the time after cell passage at which cells are exposed to electric pulses affects GET efficiency. The GET and MFI of all plasmid concentrations differed significantly when comparing cells transfected 24 h and 48 h after passage.