Comparative Analysis of Non-viral Transfection Methods in Mouse Embryonic Fibroblast Cells

Abstract

Mouse embryonic fibroblast (MEF) cells are an important in vitro model for developmental biology, disease, and reprogramming studies. However, as with other primary cells, they are challenging to transfect. Although viral gene-delivery methods achieve high gene-delivery efficiency, challenges with cell mutagenesis and safety among others have led to the use and improvement of non-viral gene-delivery methods in MEF cells. Despite the importance of gene delivery in MEF cells, there is limited comparison of method/reagent efficacy. In this study, we compared the effectiveness of different gene-delivery methods and several reagents currently available in MEF cells by introducing a plasmid containing enhanced green fluorescent protein (EGFP). We analyze transfection efficiency by EGFP fluorescence. Our results suggest that two gene-delivery methods-electroporation and magnetofection in combination with a lipid reagent, are the most efficient transfection methods in MEF cells. This study provides a foundation for the selection of transfection methods or reagents when using MEF cells.

Keywords: MEF; dendrimer; electroporation; lipid-based transfection; magnetofection.

FIGURE 1.

Transfection efficiencies by different reagents in MEF cells. Transfection efficiency was calculated as described in Materials and Methods and represented as histograms. (A and B) For TransFectin and Lipofectamine, different amounts of DNA plasmid (0.6, 0.8, and 1 μg) were combined with 2 μl reagent. Bars represent the means ± sd of 6 cell field photographs at 44 h. Student's t test showed not significant differences. For TransFectin, 0.6 vs. 0.8 μg (P = 0.6), 0.6 vs. 1 μg (P = 0.4), and 0.8 vs. 1 μg (P = 0.5). For Lipofectamine, 0.6 vs. 0.8 μg (P = 0.1), 0.6 vs. 1 μg (P = 0.5), and 0.8 vs. 1 μg (P = 0.5). Average number of cells counted per histogram bar is 674 for TransFectin and 461 for Lipofectamine. (C) For GenJet, different times of incubation of the transfection reagent were tested (5, 12, and 18 h). Bars represent the means ± sd of 6 cell field photographs at 44 h. Student’s t test: 5 vs. 12 h (P = 0.0007), 5 vs. 18 h (P = 0.001), and 12 vs. 18 h (P = 0.4). Average number of cells counted per histogram bar is 671. (D) For DreamFect Gold, different amounts of DNA plasmid (0.6, 0.8, and 1 μg) and different times of transfection incubation time (5 and 24 h) were tested. Bars represent the means ± sd of 6 cell field photographs at 44 h. Student’s t test: at 5 h, 0.6 vs. 2 μg (P = 0.04). No other statistically significant differences were found. Average number of cells counted per histogram bar is 432. (E) For NanoJuice, we combined various amounts of NanoJuice Core Transfection (0.8 and 1.6 μl) and NanoJuice Transfection Booster (0.8, 1.6, 2.4, and 3.2 μl). Bars represent the means ± sd of 4 cell field photographs at 21 h. Student’s t test: 0.8/0.8 vs. 0.8/3.2 (P = 0.02), 0.8/1.6 vs. 0.8/3.2 (P = 0.01), 1.6/0.8 vs. 0.8/2.4 (P = 0.03), and 1.6/0.8 vs. 0.8/3.2 (P = 0.003). Average number of cells counted per histogram bar is 180. (F) For GeneJuice, we tested DNA plasmid amount (0.5, 0.75, 1, 1.25, and 1.5 μg) in combination with 0.75 μl transfection reagent. Bars represent the means ± sd of 4 cell field photographs at 21 h. Student’s t test: control vs. 1.25 (P = 0.02) and control vs. 1.5 (P = 0.04). Average number of cells counted per histogram bar is 161. (G) For electroporation, 4 different pulse magnitudes (250, 300, 325, and 350 V) were tested. Bars represent the means ± sd of 4 cell field photographs at 21 h. Student’s t test: 250 vs. 325 V (P = 0.0006), 250 vs. 350 V (P = 0.006), and 300 vs. 325 V (P = 0.02). Average number of cells counted per histogram bar is 72.

FIGURE 2.

CombiMag increases the transfection efficiency of lipid reagents in MEF cells. Different amounts of CombiMag [0 (control), 0.5, 1, and 2 μl] were added to the different transfection reagents. Histograms represent the effect of CombiMag on the transfection efficiencies of lipid reagents in MEF cells at 22 h. Bars represent the means ± sd of 4 cell fields. Student’s t test: *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.00005. Average number of cells counted was 200.

FIGURE 3.

EGFP fluorescence in transfected MEF cells, which were examined 22 h after transfection of an EGFP-expressing plasmid construct by fluorescence microscopy. Panels show bright-field (grayscale) and corresponding UV images (color) for each reagent. (A) TransFectin, (B) Lipofectamine, (C) GenJet, (D) DreamFect Gold, (E) NanoJuice, (F) GeneJuice, (G) Electroporation, and (H) control transfection with plasmid vector alone. Photographs were taken at 10× original magnification. Original scale bars, 100 μm.