# Nucleofection as an Efficient Method for Alpha TC1-6 Cell Line Transfection
## 3 Results and Discussion  
### PEI-Based Transfection of \(\alpha\)TCI-6 Cells  
The rapidly developed genome engineering technologies, such as CRISPR/Cas9, offer many possibilities for application in research. For efficient (epi)genome editing, the imperative factor is efficient delivery in the host cells. Transfection is a widely used method for the delivery of foreign nucleic acid or protein in eukaryotic cells which allows for studying the regulation of gene expression and the function of gene products by enhancing or inhibiting its expression [9]. As each mammalian cell type has a specific set of requirements for efficient transfection, there is a tremendous need to optimize the protocol for cell transfection. Depending on the effect that the epigenetic manipulation in the cell expects to ensure, there is a need for a time course plan of the experiment. In this research, the mouse pancreatic cell line \(\alpha\)TCI-6 was used as a model system for epigenetic manipulation. This cell line is difficult to propagate, cells are sensitive to rough manipulation and oxygen level. Our main goal was to find a satisfying transfection condition that will provide a sufficient mass of transfected cells at a few points during the time course.  
One of the widely used methods for introducing foreign DNA into the host cell is transfection by using PEI [22]. This type of chemical transfection is considered the gold standard because PEI is powerful, easy to use, quick, cost-effective, and shows low cytotoxicity. This cationic polymer condenses DNA into stable, positively charged particles that bind to anionic cell surfaces where they have been endocytosed and released in the cell cytoplasm [23]. For the first step, small reporter plasmid mVenus C1 was used, and the most common PEI:DNA ratio (3:1) to examine transfection efficiency (Figure 1). Cells were propagated until a fifth dpt, and no negative effects on cell viability and proliferation were observed. Transfected cells behaved and looked equally good as untransfected cells. Moreover, they showed a statistically higher number of cells, indicating a better proliferation rate compared to the control untransfected cells (Figure 1A). The GFP expression level was measured as a marker for transfection efficiency [24]. Flow cytometry showed \(\sim\)30% GFP positive cells which was encouraging regarding untouched cell viability (Figure 1B,C). Moreover, cells were visualized by fluorescent microscopy on the fifth dpt to check the retention of plasmid DNA through cell division (Figure 1D). This level of transfection efficiency was satisfying, so for the next step, cells were transfected with an EpiCRISPR plasmid mix.  
In the next step, cells were co-transfected with a mix of plasmids: 5% mVVenus C1 (as a reporter gene), 20% dCas9-Dnmt3a3L-KRAB plasmid (as effector domain), 75% empty gRNA plasmid (in this study, the plasmid lacked the information for targeted genome region but in the future, it will carry the designed sequence for gRNA) with the same PEI:DNA ratio as previously described. The same transfection reagent, PEI, was used by for transfection of the FACE plasmid in different mammalian cell lines with high transfection rate and cell viability upon transfection [25]. On the fifth dpt, the flow cytometer and cell sorter were used for measuring and collecting transfected GFP positive cells. As proof of EpiCRISPR system delivery, the relative level of Cas9 mRNA was measured in the sorted cell fraction (Figure 2C). The detected level of GFP positive cells by fluorescent microscopy and FACS (Figure 2A,B) was very low (around 3.8%) relative to previous results (Figure 1). The potential reason for weak transfection efficiency could be the large size of the EpiCRISPR plasmid which is 11,548 bp. The same assumption was drawn by [PERSON], et al. in 2016, who showed that transfection efficiency is inversely correlated to the plasmid size 24 h after plasmid electrotransfer in adipose tissue-derived mesenchymal stem cells [21].