# Nucleofection as an Efficient Method for Alpha TC1-6 Cell Line Transfection
## 3 Results and Discussion
### Nucleefection of \(\alpha\)TC1-6 Cells
screened several non-viral methods (Lipofectamine2000 (Thermo Fisher Scientific, Waltham, MA, USA), TurboFect (Thermo Fisher Scientific, Waltham, MA, USA), GeneJuice (Sigma-Aldrich, St. Louis, MO, USA) and electroporation (Thermo Fisher Scientific, Waltham, MA, USA)) to deliver CRISPR/Cas9 in primary myoblasts, showing that electroporation has at least 2.5\(\times\) higher transfection efficiency than other chemically based methods [35]. Electroporation performed with a Nucleofector(tm) II/2b device was selected for cotransfecting plasmids encoding all elements for CRISPR/Cas system together with piggyBac transposon system for delivery and ex  
Figure 9: The functionality of \(\alpha\)TC1-6 was not disturbed after nucleofection. (**A**) Cells were immunostained with the anti-glucagon antibody on the fifth and seventh days after transfection. Cell nuclei were stained with DAPI. (**B**) Relative expression level of Cas9 mRNA in control untransfected cells and in cells on the fifth and seventh days after transfection measured by RT-qPCR. The results are expressed as means \(\pm\) SDs. Significance between samples was determined by using one-way ANOVA followed by [PERSON]’s post hoc test. * \(p\leq 0.05\), *** \(p\leq 0.0001\) compared to untransfected cells. pression CAR gene [36]. For malignant B cells, which are extremely difficult to transfect, Nucleofector(tm) technology-based electroporation system was used to make ROR1 knockout cells by using the CRISPR/Cas9 system [37]. Recent approaches used electroporation for successful delivery of Cas9 ribonucleoprotein (RNP) complexes consisting of the Cas9 protein and a fluorescently labeled crRNA/tracrRNA duplex targeting genes [38; 39; 40]. As shown in the paper of [PERSON] et al. (2019) the CRISPR-based construct (sgRNA:dcas9-VPR) was delivered to HEK293T cells by using FuGene HD (Promega) but delicate, C6 glioma cells were transfected by using a Nucleofector 2b device (Lonza) [41]. The \(\alpha\)TC1-6 cells are adherent and grow as loosely attached clusters with some single cells in suspension. PEI-mediated cell transfection is not a suitable method for the cells growing in clusters because there is a part of the exposed cells to DNA/PEI complexes, whereas the rest of the cells are unable to be in contact with the DNA/PEI complexes due to their clustering tendency. Therefore, in our study, nucleofection was chosen as the transfection method for \(\alpha\)TC1-6 cells, because we observed that these cells are harsh to maintain in culture, are growing in clusters, and are very sensitive to the lack of CO\({}_{2}\); therefore we foresee problems with transfection, especially because our DNA construct is rather large (-11 kb). Regarding the transfection efficiency, an approximately 40% nucleofection efficiency was observed earlier for the \(\alpha\)TC1-6 cell line transfected with the pCMV-3 FLAG-3a plasmid carrying the cloned gene upstream (5\({}^{\prime}\)) of the triple-FLAG repeat moiety. In this experiment, nucleofection was performed on an Amaxa 2b device [42]. After optimization, we succeeded to achieve approximately 70% nucleofection with the Amaxa 4D X unit. Finally, one of the reasons for better efficiency of nucleoporation could relay in the use of cell suspension whereby all cells have an equal chance to be transfected. All of this together emphasizes nucleofection as a major technique for the efficient delivery of EpiCRISPR in vitro in \(\alpha\)TC1-6 cells. Conceptualization, M.V. and J.A.J. ; methodology, M.D., M.J.T., and V.P. ; validation, M.D. and J.R.; formal analysis, M.D., A.T., and A.U. ; investigation, M.D. and M.M. ; writing--original draft preparation, M.D. ; writing--review and editing, M.V. and J.A.J. ; visualization, M.D., N.G., and S.D. ; supervision, J.A.J. ; funding acquisition, M.V. and I.M. All authors have read and agreed to the published version of the manuscript.