Systemic Optimization of Gene Electrotransfer Protocol Using Hard-to-Transfect UT-7 Cell Line as a Model

Abstract

Non-adherent cells are difficult to transfect with chemical-mediated delivery methods. Electroporation is an attractive strategy to transfer the molecules of interest into suspension cells. Care must be taken with the viability of the transfected cells since parameters, which increase cell membrane permeability, subsequently increase transfection efficiency, leading to higher cell death indices. We intended to evaluate the distribution of hard-to-transfect UT-7 cells among different subpopulations: transfected/viable, untransfected/viable, transfected/dead, and untransfected/dead populations, for a better understanding of the relation between gene electrotransfer efficacy and cell death. The following electroporation parameters were tested: pulse strength, duration, plasmid DNA concentration, and ZnSO4 as DNase inhibitor. BTX T820 square-wave generator was used, and 48 h after electroporation, cells were observed for viability and fluorescence analysis. Increasing pulse strength correlated directly with an increased ratio of pEGFP-positive cells and inversely with cell viability. The best results, representing 21% pEGFP positive/viable cells, were obtained after EP with 1 HV 1400 V/cm pulse of 250 µs duration using 200 µg/mL plasmid concentration. Results demonstrated that plasmid concentration played the most significant role in pEGFP electrotransfer into UT-7 cells. These results can represent a relevant improvement of gene electrotransfer to obtain genetically modified suspension cells for further downstream experiments.

Interplay between Electric Field Strength and Number of Short-Duration Pulses for Efficient Gene Electrotransfer

“…Gene electrotransfer is a nonviral method for gene transfer into cells and tissues that exploits short high voltage electric pulses. The interest in this method's applicability increased with time after proving GET efficacy, especially on nonadherent hard-to-transfect cells and tissues [10,12,15,25,34]. Most of the gene electrotransfer protocols utilize long pulses that are in the millisecond range (2-20 ms) [17,35]; differently, ESOPE protocol exploits short 100 µs pulses for electrochemotherapy to boost the uptake of chemotherapeutic medications [32,36].…”

“…Studies have attempted to improve the efficiency of gene electrotransfer by modifying electric field settings, pulsing buffers, plasmid concentrations, and the shape and material of the electrodes [21][22][23]. However, optimization of the electric field parameters and plasmid concentration has been found to be the most effective for improving gene electrotransfer efficiency [24,25]. Studies have demonstrated that factors such as the duration of the squarewave pulse, the quantity of pulses, the strength of the field, and the frequency of pulse application can collectively impact gene delivery through electroporation [26][27][28].…”