Prior to the advent of transfection lipids, mouse fibroblast NIH 3T3 cells were essentially untransfectable and other methods such as retroviral gene delivery had to be employed to circumvent this problem. Although retroviruses can be used for transgene expression in 100% of cells, the expression is often low. Additionally, NIH 3T3 cells are traditionally used for cellular transformation studies, and creating oncogenic retroviruses is not ideal due to bio-safety concerns. Recently, lentivruses have been used for high gene expression in cells (up to 100%) . However, this method causes even greater bio-safety concerns and requires updating culture facilities to bio-safety level 2+, resulting in a costly and time-consuming procedure. Therefore, it is of great interest to determine the best lipid reagent for transfecting NIH 3T3 cells so that cellular transformation studies can be done without updating one's facilities or creating oncogenic viruses [2,3]. Five different lipid reagents from three separate companies that claim their reagents work well in NIH 3T3 cells were compared for cytotoxicity, transfection efficiency, and optimization burden.
Materials and Methods
NIH 3T3 cells (ATCC® CRL-1658™) and HA-RAS transformed NIH3T3 cells were seeded to a density of 25,000 cells per well of a 24-well dish on coverslips in quadruple. After 24 hours, cells were transfected with 0.5 µg EGFP plasmid. Transfection efficiency was determined 48 hours later by counting GFP-expressing cells.
Cells were transfected with the following transfection reagents using manufacturers' instructions and all manufacturer-recommended optimization parameters: FuGENE® 6 Transfection Reagent, FuGENE® HD Transfection Reagent, transfection reagent LT-1, transfection reagent LX, and transfection reagent LX + PLUS.
Transfection efficiency and cytotoxicity
Percent cells transfected was measured by counting the number of cells expressing GFP in each field of a fluorescent microscope and dividing by the total number of cells. This was performed five times per well, and the average was taken as the percent transfected for that well. Cells were then fixed with 4% paraformaldehyde and mounted on a slide for visualization and quantification using a Leica SP2 microscope and Imaris software (Bitplane AG, Switzerland). Cytotoxicity was assessed by determining the number of cells alive 48 hours post transfection compared with untransfected cells.
Optimization was easy and required one simple round of experiments for all reagents. Transfection reagent LT-1 showed minimal difference between all parameters tested, and optimization does not seem to change its cytotoxicity or transfection efficiency, whereas the others tested showed variation between all the conditions tested. The percentage of tranfection is summarized in Table 1. Notably, the procedure for transfection reagent LX requires a medium change after incubation with the lipid-DNA mixture, as otherwise cytotoxicty greater than 50% will occur (data not shown), while the other tranfection reagents did not require any further handling steps until transgene expression efficiency was measured.
Only transfection reagent LX and transfection reagent LX + PLUS showed cytotoxicity, while the others showed minimal to no cell death even after prolonged incubation with lipid-DNA mixtures (Table 1).
All five lipid reagents were compared under optimized conditions for percent of cells that expressed GFP as an indicator of transfection efficiency. FuGENE® 6 Transfection Reagent and FuGENE® HD Transfection Reagent gave the greatest percentage of transfection in untransformed NIH 3T3 cells, while FuGENE® HD Transfection Reagent outperformed all the others in transformed NIH 3T3 cells (Table 1, Figures 1 and 2). Notably, in transformed cells all the reagents except transfection reagent LX were able to transfect 60% of cells, although the transfection reagent LX + PLUS caused immense cell death (Table 1). In untransformed cells, however, FuGENE® 6 Transfection Reagent and FuGENE® HD Transfection Reagent greatly outperformed the other tranfection reagents, which had only negligible transfection efficiency. Interestingly, all reagents were able to transfect transformed cells with a much greater efficiency than wild-type cells.
The Transfection Reagent LX is able to transfect cells to levels compatible with in vivo studies; however, it is also cytotoxic and should not be employed in studies where healthy NIH 3T3 cells are required. This reagent also requires the greatest time expenditure for transfection and optimization. FuGENE® 6 Transfection Reagent and transfection reagent LT-1 perform very similar in all parameters tested, though transfection reagent LT-1 is more consistent across optimization parameters while FuGENE® 6 Transfection Reagent has slightly better transfection efficiencies in untransformed cells. FuGENE® HD Transfection Reagent performs the best in percent cells transfected in both transformed and untransformed NIH 3T3 cells, has no cytotoxicity, and is as easy to use and optimize as all the others. Taken together, these facts show that FuGENE® HD Transfection Reagent is the best reagent tested for transient transfection of NIH 3T3 cells with lipids under manufacturer-recommended conditions.
1. Kafri T (2004) Methods Mol Biol 246:367–390
2. Azzam T, Domb AJ (2004) Curr Drug Deliv 1:165–193
3. Tranchant I et al. (2004) J Gene Med 6 [Suppl 1]:S24–S35
Fugene is a registered trademark of Fugent, L.L.C., USA.
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This article was originally published in Biochemica 4/2007, pages 18-19. ©Springer Medizin Verlag 2007