# Nucleofection as an Efficient Method for Alpha TC1-6 Cell Line Transfection
## 3 Results and Discussion
### Nucleefection of \(\alpha\)TC1-6 Cells
**(A)** Cell viability was observed by flow cytometry and cell viability at cell density of 2 \(\times\) 10\({}^{5}\) has been taken as a 100%. The results are expressed as means \(\pm\) SDs. Significance between groups was determined by using one-way ANOVA followed by [PERSON]’s post hoc test, * \(p\)\(\leq\) 0.05 compared to all three groups of cells. **(B)** Cell viability and **(C)** the percent of GFP-positive cells are measured by flow cytometry. For monitoring nucleofection efficiency, 3.5 \(\times\) 10\({}^{5}\) cells were transfected with 0.5 \(\upmu\)g of plasmid DNA pmaxGFP(tm) by using the two most potent predefined programs for nucleofection (CM-137 and CM-150). The results are expressed as means \(\pm\) SDs. Significance between samples was determined by using an unpaired Students \(t\)-test, * \(p\)\(\leq\) 0.05, ** \(p\)\(\leq\) 0.01.  
dCas9-Dnmt3a3L-KRAB plasmid leading to improved transfection efficiency [30]. The drop in transfection efficiency is much slighter after nucleofection compared to the DNA:PEI transfection method (Figure 2A) because the size of the plasmid has a lesser impact on transfection efficiency when a larger plasmid is used in combination with a small one (as it is in our study). Moreover, the increase in the total amount of plasmid DNA leads to twice the transfection efficiency (Figure 6B). Program CM-150 was selected for further optimization experiments because earlier it showed better cell viability after nucleofection with pmaxGFP(tm) DNA plasmid relative to CM-137 (Figure 5B,C). Although we observed improved DNA transfection efficiency with the nuclear transfer of plasmids by nucleofection in comparison with DNA:PEI transfection, fine-tuning of the selected pulse was done to further increase transfection efficiency (Figure 7). For this experiment, we used 2 ug (mix of plasmids: 5% pmaxGFP(tm), 20% dCas9-Dnmt3a3L-KRAB plasmid, 75% empty gRNA plasmid) of plasmid DNA and transfected 5 \(\times\) 105 \(\alpha\)TC1-6 cells suspended in 20 \(\mu\)L SF 4D-Nucleofector X Solutions. The program CM-156, which is designed for higher efficiency, was selected from the fine-tuning matrix (Figure 7C) and compared with CM-150 in an attempt to increase the number of GFP positive cells. On the second day after transfection, cells were observed by fluorescent microscopy (Figure 7A). On the fifth day after transfection was chosen as the next time point, because it is an optimal time frame for the EpiCRISPR system to make necessary changes in the epigenome and for those changes to become measurable [31]. An increase in the percentage of transfected cells (Figure 7B), if compared to the previous experiment after nucleofection using the CM-150 program (Figure 6B), could be a consequence of the doubling of plasmid DNA (from 1 to 2 ug) as well as increasing the cell number per transfection (from \(3.5\times 10^{5}\) to \(5\times 10^{5}\)). Presented results indicated that the CM-156 program provided an improved effect on transfection efficiency as can be seen by the amount of GFP positive cells with a limited effect on cell growth. For transient transfection, introduced genetic material is not integrated into the genome and is expressed only for a restricted period, because it can be lost by environmental factors and cell division [19]. The optimal time point for measuring the effect of introduced DNA into the nucleus of the cells is between the first or fourth dpt [9]. However, when transfection serves for delivery of the epigenome-editing machinery into the cells, effects of introduced epigenetic changes like DNA methylation, could require more time to make the physiological effects. The selected program CM-156 was tried in large-scale cuvettes, and the results were scaled and reproducible (Figure 8). For this experiment, cells were nucleofected in 100-\(\mu\)L Nucleovette(tm) vessels with a 7.5 ug of plasmids mix (5% pmaxGFP(tm), 20% dCas9-Dnmt3a3L-KRAB plasmid, 75% empty gRNA plasmid). We  
Figure 6: Optimization of the selected programs and DNA amount for nucleofection with EpiCRISPR plasmids.