Transfection of insect cell lines using polyethylenimine

Abstract

Insect cell lines have been widely used in recombinant baculovirus expression systems and transient gene expression studies. Critical to these applications have been the transfection of foreign DNA. This has been frequently done using labor intensive and cytotoxic liposome-based transfection reagents. In the current study we have optimized a new kind of polyethylenimine-based DNA transfection reagent on the Spodoptera frugiperda Sf9 insect cell line. A plasmid vector that transiently expresses green fluorescent protein (GFP) was effectively delivered into Sf9 cells. A transfection efficiency of 54% and cell viability of 85-90% were obtained for Sf9 cells. The developed transfection protocol has now been successfully used to transfect eight insect cell lines derived from Bombyx mori, Trichoplusia ni, Helicoverpa zea, Heliothis virescens and S. frugiperda with GFP and GUS with transfection efficiencies of at least 45%. This method provides high heterologous protein expression levels, transfection efficacy and cell viability, and could be used for transient gene expression in other lepidopteran cell lines.

Fig. 1

Imaging of GFP-specific fluorescence. (A) Sf9 cell were transfected with the pBAC-5-GFP plasmid and visualized at 72 h post transfection using an inverted fluorescent microscope and GFP S filter system under UV-light. For transfection were used 0.5 μg (i), 1.0 μg (ii) and 2.0 μg (iii) of plasmid DNA and 6 eq of transfection reagent ExGen500. Mock transfected cells (M) also included and represent Sf9 cells transfected with plasmid DNA in the absence of ExGen500. (B) The same Sf9 cells were scanned for GFP-specific fluorescence using Typhoon 9,200 variable mode imaging system at the resolution of 25 μm. Each well is 16 mm in diameter. For transfection were used the following ratios of DNA:ExGen500: wells 1, 2, 3–0.5 μg plasmid DNA and 6 eq (1), 8 eq (2), 10 eq (3) ExGen500; wells 4, 5, 6–1.0 μg plasmid DNA and 6 eq (1), 8 eq (2), 10 eq (3) ExGen500; wells 7, 8, 9–2.0 μg plasmid DNA and 6 eq (1), 8 eq (2), 10 eq (3) ExGen500; wells 10, 11, 12-mock, Sf9 cells transfected with plasmid DNA in the absence of ExGen500 (10), Sf9 cells transfected in the absence of plasmid DNA (11), mock Sf9 cells

Fig. 2

Influence of different DNA:ExGen500 ratios on the efficiency of transfection (A), cellular expression level (B) and viability of Sf9 cells (C). (A) Sf9 cells were transfected using ExGen500 at the different DNA:ExGen500 ratios. At 72 h post transfection the percentage of transfected GFP-positive cells was determined. Experiments were done in triplicate. The error bars represent standard error. (B) For determination of cellular expression levels of transfected cells measured as pixel densities or rfu/pixel², transfected Sf9 cell were scanned for GFP-specific fluorescence using Typhoon 9,200 variable mode imaging system. Experiments were done in triplicate and the results were measured as pixel densities or rfu/pixel². The error bars represent standard error. (C) Toxicity of ExGen500 reagent was evaluated by Trypan Blue exclusion. Experiments were done in triplicate