An improved method for increasing the efficiency of gene transfection and transduction

Abstract

Transfection and transduction using lentivirus has gained attention in biomedical research. To date, how to reach the maximum transfection and viral transduction efficiency is still challenging. Here we compared the transfection and viral transduction efficiency using commercially available transfection reagents including FuGENE 6, Lipofectamine 2000 and Lipofectamine 3000 in different cell lines and primary cultured cells. Enhanced green fluorescent protein (EGFP) was clearly seen in Eppendorf tubes from harvested cells using Lipofectamine 3000 without using a microscope and UV activation. Strong expression of EGFP was observed in HEK293 cells, mouse primary cortical neurons and human umbilical vein endothelial cells (HUVECs) using confocal microscopy. Western blot showed the strongest EGFP expression using cell lysates from Lipofectamine 3000 transfected HEK293 cells and transduced HUVECs compared with Lipofectamine 2000 or FuGENE 6 reagents. Using Cx43 shRNA lentivirus combined with Lipofectamine 3000 transfection reagent, we can achieve about 90% Cx43 knockdown efficacy in HUVECs. Therefore, our results suggest that a much higher transfection and viral transduction efficiency can be attained by using Lipofectamine 3000 transfection reagent.

Keywords: EGFP; Lipofectamine 3000; Transfection; connexin43; viral transduction.

Figure 1

Comparison of transfection efficiency after transiently transfected lentivirus expression vector plenti CMV GFP DEST GFP into HEK293 cells using Lipofectamine 3000, 2000 or FuGENE 6 reagents. Two days after transfection, the GFP in living cells was monitored using the same lower power setting with 4× objective lens in an Olympus confocal microscope. It showed the highest GFP expression using Lipofectamine 3000 transfection reagent (A) as compared with using Lipofectamine 2000 (B) or FuGENE 6 (C) transfection reagents.

Figure 2

Comparison of viral transduction efficiency in HUVECs using lentivirus generated from HEK293 cells utilizing Lipofectamine 3000, 2000 or FuGENE 6 reagents. Three days after viral transduction, the GFP in living cells was monitored using the same lower power setting with 10× objective lens in an Olympus confocal microscope. It showed the highest GFP expression using lentivirus generated with Lipofectamine 3000 transfection reagent (A) as compared with using Lipofectamine 2000 (B) or FuGENE 6 (C) transfection reagents.

Figure 3

Comparison of viral transduction efficiency in mouse cortical neurons using lentivirus generated from HEK293 cells utilizing Lipofectamine 3000, 2000 or FuGENE 6 reagents. Three days after viral transduction, the GFP in living cells was monitored using the same lower power setting with 10× objective lens in an Olympus confocal microscope. It showed the highest GFP expression using lentivirus generated with Lipofectamine 3000 transfection reagent (A) as compared with using Lipofectamine 2000 (B) or FuGENE 6 (C) transfection reagents.

Figure 4

Without using a fluorescence microscope and without UV activation, GFP was clearly seen in harvested cells after 2 days of transfection in HEK293 cells using Lipofectamine 3000 or three days of viral transduction in HUVECs using lentivirus generated from HEK293 cells utilizing Lipofectamine 3000 reagent. Two days after transfection in HEK293 cells using Lipofectamine 3000 or three days after viral transduction in HUVECs with lentivirus generated from HEK293 cells utilizing Lipofectamine 3000 reagent, the cells were harvested, and GFP was clearly in these harvested HEK293 cells (A, tube 1) and HUVECs (B, tube 1) even without using a confocal microscope or without using UV activation. On the contrary, we can not see any obvious GFP fluorescence under the same condition but using Lipofectamine 2000 (A and B, tube 2), or FuGENE 6 (A and B, tube 3) or non-transfection or non-transduction controls (A and B, tube 4).

Figure 5

Western blot showing high level of GFP expression in cortical neuron cell lysates after viral transduction using Lipofectamine 3000 reagent. Using cell lysates from HUVECs after three days of viral transduction with virus generated utilizing Lipofectamine 3000 reagent, western blot showed a strong GFP band migrating from the top of the gel to the bottom after using 10 ug of total protein loading, however, there is absent of GFP detection using cell lysates without viral transduction (D). After SDS-PAGE using 1 ug protein loading, a single protein band migrating around 25 kDa is clearly seen using cell lysates from virus transduced mouse cortical neurons (A, upper panel, lane 1), but the band is absent using cell lysates from mouse cortical neurons without undertaking viral transduction or using supernatant of lentivirus only (A, upper panel, lane 2 and 3), the same membrane was striped and reprobed with anti-beta actin antibody showed equal beta-actin detection (A, lower panel, lane 1 and 2), but there was no beta-actin band detection using supernatant containing lentivirus only (A, lower panel, lane 3). Western blot also showed highest GFP protein expression in mouse cortical neurons using virus generated from HEK293 cells transfected with Lipofectamine 3000 reagent (B, upper panel, lane 3), while less GFP is detected using virus produced with FuGENE 6 or Lipofectamine 2000 transfection reagents (B, upper panel, lane 1 and 2), the same membrane was striped and reprobed with anti-beta actin antibody, which showed equal beta-actin detection (B, lower panel). Similarly, statistics analysis also showed that GFP protein level using lipofectaime 3000 reagent is at least three times higher than using Lipofectamine 2000 or FuGENE 6 transfection reagents (C).

Figure 6

High efficiency of Cx43 knockdown in HUVECs using lentivirus generated from HEK293 cells utilizing Lipofectamine 3000 transfection reagent. Immunofluorescence labeling of Cx43 in HUVECs showed significantly reduced Cx43 punctate labeling using the lentivirus generated utilizing Cx43 shRNA plasmids combined with Lipofectamine 3000 reagent (C) compared with Cx43 labeling in HUVECs using Cx43 shRNA combined with Lipofectamine 2000 transfection reagent (B) or using scrambled Cx43 shRNA plasmids with Lipofectamine 3000 reagent (A). The same result was also verified using western blot (D), there was statistically significant reduced Cx43 detection in HUVECs cells using lentivirus produced with Cx43 ShRNA and Lipofectamine 3000 transfection reagents, which showed about 90% Cx43 knocked down, compared with about 60% Cx43 knockdown using lentivirus generated utilizing Cx43 ShRNA plasmids and Lipofectamine 2000 transfection reagent (E).