# Efficient Gene Transfection by Electroporation--In Vitro and In Silico Study of Pulse Parameters
## 4 Discussion
### Effect of Pulse Parameters on Overall Gene Electrotransfer  
We achieved GET in both cell lines with all pulse protocols, but with variable efficiency (Figure 8). Differences in GET efficiency between different cell lines have been reported before [86, 87, 88] and are still not well understood. It has been suggested that differences in GET efficiency can be the consequence of various biophysical factors such as the fluidity of the cell membrane or biological parameters such as different mechanisms that are present or activated in cells and the ability of cells to recover after the delivery of electric pulses [89, 90]. If the difference would be the consequence of different cell membrane composition or fluidity or induced transmembrane voltage, then we would expect to also observe the difference in cell membrane permeabilization. Since permeabilization and survival curves were similar for both cell lines, the difference in GET efficiency is more likely the consequence of the difference in the presence and degree of activity of intrinsic cellular mechanisms and pathways. Furthermore, in in vitro experiments, the composition of the electroporation medium can also influence GET efficiency [91, 92, 93]. The GET of each cell line in our experiments was performed in their recommended growth medium. For C2C12 myoblasts, this was DMEM, and for 1306 fibroblasts, this was EMEM. They are both variations of basic medium used for primary and diploid cultures, but DMEM has a higher concentration of amino acids and vitamins compared to EMEM. DMEM also contains iron in the form of ferric sulfate, which is absent in EMEM [94].  
We performed GET in growth medium, which is more like in vivo extracellular fluid compared to other buffers used for GET. Growth medium is highly conductive, which results in high currents and increased electrochemical reactions such as electrolysis, the generation of radicals, and the release of metal ions from the electrodes during pulse delivery [28, 95]. It was shown previously that short, i.e., nanosecond, pulses and bipolar pulses decrease metal release from electrodes compared to longer pulses [29, 35, 48, 96]. Furthermore, high medium conductivity also results in high currents, leading to heatingand temperature increases in the sample during pulse delivery. The temperature increase is more pronounced when longer or more pulses are applied [35]. In our experiments, the temperature of the sample never exceeded 32 \({}^{\circ}\)C, meaning that thermal effects were negligible (Supplementary Table S5).  
In addition to achieving a higher percentage of transfected cells in 1306 fibroblasts, the MFI of the GFP-positive cells was higher (Figure 9). Fluorescence intensity is reported to be dependent on the number of pDNA copies inside the cell that have reached the cell nucleus and have been successfully transcribed and translated into fluorescent proteins [97; 98]. In this respect, this could mean that the higher number of pDNA copies was transferred to 1306 fibroblasts during pulse delivery compared to C2C12 myoblasts. Other factors, such as the availability and degree of activity of the cellular machinery for transcription and translation, also affect production of proteins from transfected pDNA [97; 98]. Based on this, higher GET efficiency in 1306 fibroblasts could also mean that the rate of pDNA transcription and translation into fluorescent protein is higher in 1306 fibroblasts compared to C2C12 myoblasts.