Many viruses, including human cytomegalovirus (HCMV), encode for genes that can modulate or interfere with the ability of cells to mount an effective antiviral response to infection [1, 2]. Transfection of viral genes into cultures of mammalian cells is an important research tool to evaluate the function of viral gene products. We utilized this approach to identify viral genes involved in modulation of interferon (IFN)-mediated cell signaling pathways. However, our efforts were seriously hampered by activation of cellular interferon responses caused by the transfection procedure. Induction of IFN-stimulated genes by transfection of nucleic acids may not only lead to misinterpretation of results derived from cell culture studies, but may also be a cause for concern for gene delivery in vivo.
In an attempt to overcome this problem, we analyzed expression of a reporter gene, CD2, under the control of a promoter element derived from an interferon-stimulated gene (ISG), following transfection of plasmid DNA with several commercially available transfection reagents. Here we report that only transfection of plasmid DNA with the Fugene® HD Transfection Reagent minimized non-specific induction of our reporter gene while achieving transfection efficiencies similar to those of other reagents tested in this system. We also show that induction of CD2 expression by other commercially available reagents was, at least in part, mediated by induction of IFN-ß. The Fugene® HD Transfection Reagent is thus superior to other reagents in minimizing the cellular response to transfection of plasmid DNA.
Materials and Methods
A human fibrosarcoma cell line, 2C4 (Gift of G.R. Stark), stably transfected with the T-cell surface antigen CD2 under the control of an interferon inducible promoter, was used for all experiments . The cells were maintained in DMEM (Dulbecco’s Modified Eagle Medium) supplemented with 10% fetal bovine serum, l-glutamine and sodium pyruvate.
2C4 cells (40,000 cells per well) were seeded in 12-well plates. After 24 hours in culture, cells were exposed to pEGFP-N3 plasmid DNA (Clonetech) mixed with one of three transfection reagents (Fugene® HD Transfection Reagent, transfection reagent T and transfection re-agent G) according to the manufacturers’ protocols. The DNA:transfection reagent ratios used for Fugene® HD Transfection Reagent, transfection reagent T and transfection reagent G were 1:1.5, 1:3 and 1:3 respectively. Unless otherwise specified, 1 µg of DNA per well was used in transfections with Fugene® HD Transfection Reagent and transfection reagent T and 1.75 µg of DNA was used with transfection reagent G.
Interferon treatment and flow cytometry
At 24 hours after transfection, the medium was removed and replaced with fresh medium or fresh medium containing 100 U/mL of IFN-ß (Chemicon) or neutralizing antibodies to IFN-ß (PBL Biomedical Laboratories). At 24 hours after IFN treatment, or at 48 hours after transfection, cells were dislodged from the plates by non-enzymatic treatment with versene, and resuspended in 2 ml phosphate buffered saline (PBS) with 1% newborn calf serum (NCS) in polystyrene tubes. From this point, cells were maintained at 4°C. After a second rinse in PBS 1% NCS, the cells were reacted with phycoerythrin (PE)-conjugated anti-CD2 antibody (Dako) according to the manufacturer’s recommendation. The cells were rinsed again in PBS 1% NCS, and cell surface CD2 expression (PE, FL2 channel) and the GFP expression (FL1 channel) were analyzed by flow cytometry (FACSCalibur, Becton Dickinson). The data were analyzed with the aid of CellQuest Software (Becton Dickinson).
Results and Discussion
We investigated the effect of three different transfection reagents on nonspecific induction of CD2 expression in 2C4 cells. The 2C4 fibrosarcoma cell line used in these studies harbors the CD2 gene under the control of an interferon inducible promoter. Exposure to interferons leads to an increase of CD2 antigen at the cell surface. Transfection of genes whose products modulate the interferon pathway can be evaluated by measuring CD2 expression in the presence and absence of interferon treatment. However, we observed that transfection of plasmid DNA alone led to an increase in cell surface accumulation of CD2. We therefore set out to determine if we could identify a transfection reagent that did not elicit non-specific induction of CD2 expression.
FuGENE® HD Transfection Reagent minimizes non-specific induction of CD2 expression on 2C4 cells
To evaluate expression of interferon-stimulated genes caused by transfection, 2C4 cells were cultured in 12‑well plates and transfections were performed with 1µg of pEGFP-N3 plasmid using three different transfection reagents (Fugene® HD Transfection Reagent, transfection reagent T and transfection reagent G) according to the manufacturers’ recommendation. At 48 hours after transfection, cell surface CD2 expression was measured by incubating the cells with PE-conjugated anti-CD2 antibodies and analyzing the cell populations by flow cytometry. Transfection efficiencies were measured by analyzing GFP fluorescence. As shown in Figure 1, transfection of plasmid DNA led to a discernable shift towards higher CD2 expression, reflecting increased abundance of CD2 at the surface of the cells relative to untransfected cells. The degree of CD2 accumulation at the cell surface varied according to the transfection re-agent employed in these studies (summarized in Table 1). Relative to untransfected cells, the fold increase in CD2 expression was 2.4, 4.7 and 6.6 for cells transfected using FuGENE® HD Transfection Reagent, transfection reagent T and transfection reagent G, respectively. This variability could not be explained solely by differences in transfection efficiencies as the proportion of cells in each group expressing GFP were similar (41%–46%). These data demonstrate that even in the absence of exogenously added interferons, transfection of plasmid DNA alone is sufficient to induce interferon-stimulated gene expression and that the Fugene® HD Transfection Reagent is superior in minimizing this cellular response to transfection. We also found that exposure of cells to these transfection reagents alone did not lead to upregulation of CD2 expression relative to untreated cells (Figure 2). We conclude that cells respond to the combination of plasmid DNA and transfection reagent in a manner related to the intrinsic properties of the transfection reagent.
Interestingly, we found that transfection efficiency remained high even when much lower DNA concen-trations were used in conjunction with the Fugene® HD Transfection Reagent. When cells cultured in 12-well plates were exposed to 0.25 µg of pEGFP-N3 plasmid DNA mixed with Fugene® HD Transfection Reagent, 44% of cells were positive for GFP expression. In this experiment (not shown), CD2-PE fluorescence was again minimal (1.4-fold higher than untransfected cells).
Induction of CD2 expression by transfection and IFN treatment
2C4 cells are engineered to express the CD2 antigen on their cell surface in response to IFN-ß treatment. To gauge the relative impact of transfection on expression of interferon-stimulated genes, we measured the level of cell surface CD2 expression caused by (1) transfection, (2) exposure of cells to IFN, and (3) the combination of transfection and exposure to IFN-ß. Cells were exposed to pEGFP-N3 DNA mixed with Fugene® HD Transfection Reagent, transfection reagent T and transfection reagent G (Figure 3). At 24 hours after transfection, cells were left untreated, or exposed to 100 U/mL of IFN-ß (compare panels A and B). Transfection of the 2C4 cells using the transfection reagent T and transfection reagent G, but not Fugene® HD Transfection Reagent led to sharply increased cell surface CD2 expression (compare panels A and C). In the case of transfection reagent T and transfection reagent G, the level of CD2 cell surface expression was comparable to that obtained by treating cells with 100 U/mL of IFN-ß (compare panels C and D). In fact, only in cells transfected with the FuGENE® HD Transfection Reagent did we still observe a substantial increase in CD2 expression upon IFN-ß treatment.
The non-specific induction of CD2 expression on 2C4 cells is partially blocked by anti-IFN-ß antibodies
The induction of CD2 by the transfection process could be mediated by intrinsic signaling pathways, or through induction of interferons. To test the possibility that transfection itself leads to interferon induction, 2C4 cells were exposed to pEGFP-N3 using Fugene® HD Transfection Reagent and transfection reagent G, followed immediately by addition of neutralizing antibodies to IFN-ß antibody into the culture media. Cells were harvested at 48 hours and analyzed for CD2 expression by flow cytometry (Figure 4). We observed a dose-dependent but incomplete reduction in CD2 expression with increasing amounts of neutralizing antibody in cells transfected using the transfection reagent G. Again, usage of the FuGENE® HD Transfection Reagent precluded the dramatic accumulation of cell surface CD2 which was not affected by inclusion of neutralizing antibodies to IFN-ß. Thus, induction of interferons by transfection of DNA is at least in part responsible for the expression of interferon-responsive genes.
Our results indicate that the Fugene® HD Transfection Reagent induces minimal induction of interferon-stimulated genes when used to deliver plasmid DNA. In contrast, induction of cellular response to transfection was dramatic when transfection reagent T and transfection reagent G were used. The non-specific induction of CD2 expression in 2C4 cells transfected with transfection re-agent T and transfection reagent G was comparable to that observed upon treatment with IFN-ß. The transfection-induced expression of a reporter gene under the control of an interferon-responsive promoter was not a direct consequence of transfection efficiency, nor was it due to the transfection reagents alone. Rather, plasmid DNA delivery is required to elicit this effect in a manner dependent upon the transfection reagent used. Furthermore, we found that the non-specific induction of the interferon-regulated CD2 gene in this system could, at least in part, be attributed to the induction of IFN-ß expression in the transfected cells. Therefore, the Fugene® HD Transfection Reagent is ideal for applications where non-specific induction of interferons is not desirable.
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2. Cebulla CM et al. (1999) Intervirology 42: 325–330
3. Watling D et al. (1993) Nature 366: 166–170
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This article was originally published in Biochemica 3/2006, pages 20-23. ©Springer Medizin Verlag 2006