# Efficient Gene Transfection by Electroporation--In Vitro and In Silico Study of Pulse Parameters
## 3 Results
### Effect of Different pDNA Size on GET  
Since pDNA size is reported to have an effect on GET efficiency, we performed experiments using two different size pDNA, comparing a 3.5 kb plasmid pmaxGFP and a 4.7 kb plasmid pEGFP-N1, both encoding GFP under the control of a CMV promotor. The concentrations of both plasmids in all experiments were 500 \(\upmu\)g/mL, meaning that the number of pDNA copies added was 1.34 \(\times\) higher for the smaller pDNA. We performed experiments with pmaxGFP for both cell lines for pulse protocols which gave the highest GET with pEGFP-N1 plasmid (Table 1).  
As we can observe in Figure 6A, in C2C12 myoblasts with smaller pDNA, significantly higher overall GET was observed only for 8 \(\times\) 100 \(\upmu\)s pulse protocol. Overall GET with 200 ns, 500 ns and HF-BP protocol was comparable. Interestingly, overall GET with 8 \(\times\) 5 ms was lower with smaller pDNA compared to larger pDNA.  
Similar results were obtained for 1306 fibroblasts (Figure 6B). Overall GET was significantly higher with smaller pDNA only for 8 \(\times\) 100 \(\upmu\)s protocol when compared to larger pDNA. With the 200 ns, 500 ns, HF-BP, and 8 \(\times\) 5 ms protocols, comparable overall GET was observed with both pDNA sizes.  
Figure 6: GET, cell survival, and overall GET with 500 \(\upmu\)g/mL of pmaxGFP and pEGFP-N1 pDNA with different pulse protocols for **(A)** C2C12 myoblasts, 25 \(\times\) 200 ns: 25 \(\times\) 200 ns, 10 Hz, 15.8 kV/cm; 100 \(\times\) 500 ns: 100 \(\times\) 500 ns, 10 Hz, 4.1 kV/cm; HF-BP: 2-2-2-2, 32 pulses, 100 bursts, 1 Hz, 1.25 kV/cm; 8 \(\times\) 100 \(\upmu\)s: 8 \(\times\) 100 \(\upmu\)s, 1 Hz, 1.25 kV/cm; 8 \(\times\) 5 ms: 8 \(\times\) 5 ms, 1 Hz, 0.4 kV/cm; and **(B)** 1306 fibroblasts, 25 \(\times\) 200 ns: 25 \(\times\) 200 ns 10 Hz, 12.8 kV/cm; 25 \(\times\) 500 ns: 25 \(\times\) 500 ns, 10 Hz, 6.5 kV/cm; HF-BP: 2-2-2, 32 pulses, 100 bursts, 1 Hz, 1. kV/cm; 8 \(\times\) 100 \(\upmu\)s: 8 \(\times\) 100 \(\upmu\)s, 1 Hz, 1.25 kV/cm; 8 \(\times\) 5 ms: 8 \(\times\) 5 ms, 1 Hz, 0.6 kV/cm. 0 V represents cells not exposed to electric pulses. Results are represented as an average of 3 repetitions. Bars represent standard deviation.  
When comparing MFI of all pulse protocols between different pDNA sizes a marked difference was observed between the two cell lines. In C2C12 myoblasts, the MFI of the GFP-positive cells was significantly higher with smaller pDNA (pmaxGFP) after GET with the 200 ns, 500 ns, and HF-BP protocols (Figure 7A), while 1306 fibroblasts exhibited significantly higher MFI with smaller pDNA after 8 \(\times\) 100 \(\upmu\)s and 8 \(\times\) 5 ms protocols (Figure 7B).  
Figure 7: Median fluorescence (MFI) of GFP-positive cells with 500 \(\upmu\)g/mL of pmaxGFP and pEGFP-N1 pDNA with different pulse protocols for (**A**) C2C12 myoblasts, 25 \(\times\) 200 ns: 25 \(\times\) 200 ns, 10 Hz, 15.8 kV/cm; 100 \(\times\) 500 ns: 100 \(\times\) 500 ns, 10 Hz, 4.1 kV/cm; HF-BP: 2-2-2, 32 pulses, 100 bursts, 1 Hz, 1.25 kV/cm; 8 \(\times\) 100 \(\upmu\)s: 8 \(\times\) 100 \(\upmu\)s, 1 Hz, 1.25 kV/cm; 8 \(\times\) 5 ms: 8 \(\times\) 5 ms, 1 Hz, 0.4 kV/cm; and (**B**) 1306 fibroblasts, 25 \(\times\) 200 ns: 25 \(\times\) 200 ns, 10 Hz, 12.8 kV/cm; 25 \(\times\) 500 ns: 25 \(\times\) 500 ns, 10 Hz, 6.5 kV/cm; HF-BP: 2-2-2, 32 pulses, 100 bursts, 1 Hz, 1. kV/cm; 8 \(\times\) 100 \(\upmu\)s: 8 \(\times\) 100 \(\upmu\)s, 1 Hz, 1.25 kV/cm; 8 \(\times\) 5 ms: 8 \(\times\) 5 ms, 1 Hz, 0.6 kV/cm. 0 V represents cells not exposed to electric pulses. Results are represented as an average of 3 repetitions. Bars represent standard deviation. Asterisk * represents statistically significant (\(p<\) 0.05) difference between pmaxGFP and pEGFP-N1 for the same pulse protocol.