Comparing chemical transfection, electroporation, and lentiviral vector transduction to achieve optimal transfection conditions in the Vero cell line

Abstract

Background: Transfection is an important analytical method for studying gene expression in the cellular environment. There are some barriers to efficient DNA transfection in host cells, including circumventing the plasma membrane, escaping endosomal compartmentalization, autophagy, immune sensing pathways, and translocating the nuclear envelope. Therefore, it would be very useful to introduce an optimum transfection approach to achieve a high transfection efficiency in the Vero cell line. The aim of this study was to compare various transfection techniques and introduce a highly efficient method for gene delivery in Vero cells.

Methods: In the current study, three transfection methods were used, including chemical transfection, electroporation, and lentiviral vector transduction, to obtain the optimum transfection conditions in the Vero cell line. Vero cells were cultured and transfected with chemical transfection reagents, electroporation, or HIV-1-based lentivectors under different experimental conditions. Transfection efficiency was assessed using flow cytometry and fluorescence microscopy to detect GFP-positive cells.

Results: Among the tested methods, TurboFect™ chemical transfection exhibited the highest efficiency. Optimal transfection conditions were achieved using 1 µg DNA and 4 µL TurboFect™ in 6 × 10⁴ Vero cells.

Conclusion: TurboFect™, a cationic polymer transfection reagent, demonstrated superior transfection efficiency in Vero cells compared with electroporation and lentivirus particles, and is the optimal choice for chemical transfection in the Vero cell line.

Keywords: Chemical transfection reagents; Electroporation; Lentivirus; Transfection efficiency; TurboFect; Vero cell line.

Fig. 1

GFP transfection efficiency analysis chemical-based transfection reagents. Vero cell lines were subjected to transfection using Lipofectamine™ 2000 (A), TurboFect™ (B), X-tremeGENE™ 9 (C), and PEI MAX® (D). (p ≤ 0.05 *, p ≤ 0.01**, p ≤ 0.001***, p ≤ 0.0001****)

Fig. 2

Cell viability analysis using chemical transfection reagents. Vero cell line viability was assessed using the chemical transfection reagents Lipofectamine™ 2000 (A), TurboFect™ (B), X-tremeGENE™ 9 (C), and PEI MAX® (D)

Fig. 3

GFP transfection efficiency analysis of electroporation by flow cytometry demonstrated the expression of GFP at various voltages and Ebuffer in Vero cells by flow cytometry (A), and analysis showed that there was a significant difference between Ebuffer 3 and Ebuffer 1 at 300 V (B) (p ≤ 0.01**). Comparison of cell viability in all groups with the control showed that the cell death rate was higher at 400 V than at the other voltages (C)

Fig. 4

Lentiviral vector phenotype transduction in Vero cells. GFP expression in HEK293 Lenti-X cells at 24, 48, and 72 h post-transduction (A). Flow cytometry analysis of 1,4,16,64 virus dilution (D) transduced into Vero cells (B) and transfection efficiency in all dilutions in (C)

Fig. 5

Comparison of all transfection methods. Comparison of transfection with chemical reagents showed that TurboFect had the highest transfection efficiency among other reagents in Vero cells (A). A comparison of all transfection methods showed that the use of chemical reagents, especially TurboFect, is still the most successful method for transferring DNA to Vero cells (B). (p ≤ 0.05 *, p ≤ 0.01**, p ≤ 0.001***, p ≤ 0.0001****)