Investigation of various electroporation conditions for efficient gene transfection

Gene electrotransfection (GET) is a widely used gene delivery method which is based on the application of electric fields. The phenomenon of gene delivery into cells by means of electric pulses were proved to be more complicated compared to electrotransfer of low molecular mass molecules through the cells plasma membrane and involve endocytosis. GET already demonstrated itself as a relatively inexpensive and safe method in clinical trials because plasmid DNA is introduced into the cells without viruses or additional chemicals. However, the efficiency of GET is still low when carrying large genes and cell death after GET. The aim of this research was to increase transfection efficiency without reducing cell viability. Experiments were done with Chinese hamster ovary (CHO) cell line, using different electroporation parameters to check the efficiency of GET. Green fluorescent protein (pmaxGFP) plasmid transfection efficiency was studied by measuring number of transfected cells and their fluorescence intensity with a flow cytometer (AccuriTM C6, USA) 24 h after application of different number of high voltage (HV) pulses. Cell viability was evaluated using a clonogenic assay 6 days after the experiment. It was determined that using a different number from 800 to 1600 V/cm in increments of 200 V/cm HV pulses most of the cells were transfected at field intensities of 1200- 1400 V/cm. These parameters also gave the highest cell viability and fluorescence intensity of transfected cells compared to other parameters of pulses that gave similar transfection efficiency. Our results suggest that using less number of more intensive HV pulses was more efficient for GET and preserving cell viability than using more but less intensive HV pulses. In addition, using to much intensive electric field (e.g. 1600 V/cm) was inefficient for GET, although reduced cell viability drasticallyBiochemijos katedraBiologijos katedraGamtos mokslų fakultetasVytauto Didžiojo universiteta