# Nucleofection as an Efficient Method for Alpha TC1-6 Cell Line Transfection
## 1 Introduction  
The modification of specific epigenetic signatures which define cell identity and plasticity could lead to the change of cell function without alteration in genomic DNA sequences. Powerful clustered regularly interspaced short palindromic repeats (CRISPR/Cas; CRISPR) genome-targeting technology could be redesigned for epigenome editing and targeted modulation of the gene expression (EpiCRISPR). This EpiCRISPR system contains nuclease-deactivated Cas9 (dCas9) fused with an enzyme that can change certain marks on DNA or histone tails close to the targeted DNA sequence, offering advantages over the other epigenome-editing technologies such as the simpler design for targeting new sequences [1; 2; 3]. Studies have found that combinations of several epigenetic effector domains achieve stronger effects than using only one element [4]. The pancreatic alpha cells from Langerhans islets represent a source of the hormone glucagon which is responsible for elevating blood glucose levels and maintaining glucose homeostasis. Alpha cells have reached the spotlight of scientific discovery because they serve as a protector of beta cells supporting their insulin-producing capacity [5]. Because diabetes is caused by beta cell loss, great attention is focused on alpha cells as a promisingsource for innovative diabetes therapies due to their ability to spontaneously transdifferentiate into insulin-producing cells [6]. Alpha cell hyperplasia in response to beta cell injuries offers promise as a source for increasing insulin-producing cells [7]. Because alpha cells are considered as a proper source for beta cell replacement, the development of new (epi)genetic manipulation strategies that will push alpha cells into the transdifferentiation process is needed [8]. The efficiency of introducing epigenetic modification and consequently potential biomedical application depend on the efficient delivery of foreign DNA into the host cells through the transfection process [9]. There is no ideal transfection method for different cell types originating from the same organism and especially from different sources such as plant, animal, and bacterial cells. All the transfection methods are roughly classified into viral- and non-viral-based. The non-viral transfection method can be further classified into chemical and physical/mechanical methods [10]. Each of these methods has its particular advantages and weaknesses. The method of choice depends on the experimental design and objective [9]. The chemical methods for transfection are the most widely used in research. These methods are based on the use of cationic polymer, calcium phosphate, cationic lipid, and cationic amino acid [9]. The most typical cationic polymer for delivering plasmid in vitro is polyethyleneimine (PEI). PEI efficiently induces electrostatic condensation of DNA molecules, forming positively charged nanometer-sized particles which interact with the negatively charged components of the cell membrane for endocytosis. Afterward, DNA is released into the cytoplasm [11]. PEI is broadly used because of its accessibility, high DNA delivery efficiency, and reproducibility during upscaling. DNA-PEI particle size and transfection efficiency could be affected by the many parameters, such as the amount of DNA, the DNA ratio to the PEI, the timing and the solution conditions for complex formation, the transfection medium, and cell density at the time of transfer [12]. The physical/mechanical transfection method includes electroporation, sonoporation, biolistic particle delivery, gene microinjection, and laser irradiation [13; 14], with electroporation being the most commonly used. Electroporation introduces DNA into a variety of cells by using pulsed electrical fields. Several instruments for electroporation are commercially available, and manufacturers supply guidelines for optimization of electroporation and protocols for specific cell types.