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The function of proteins can be studied in cultured cells using two distinctly different methodologies, both involving the transfection of DNA or RNA into cultured cells. The protein can be over-expressed in cells by transfecting the gene that codes for the protein, or the expression of the protein can be decreased by introduction of short segments of interfering RNA termed either siRNA (short) or miRNA (micro). In the second method, the term gene knockdown, knockout, or gene silencing is used to define the decrease in the protein of interest. X-tremeGENE siRNA Transfection Reagent has proven very effective for delivery of both siRNA and miRNA, resulting in high levels of knockdown [1–3].
Recently, FuGENE® HD Transfection Reagent has been used to deliver complete plasmids to achieve similar knockdown of proteins within cells [4–6]. Plasmids containing knockdown sequences are commercially available and are usually sold as shRNA plasmids (short hairpin) or psiRNA (this article uses the term “shRNA” to refer to these plasmids). This study was undertaken to determine if any of the parameters recommended for transfection optimization of expression plasmids would increase knockdown. Since we were not investigating the pathway used for knockdown, we decided to use shRNA plasmids known to have their effect via the siRNA pathway and to use protein expression as a measurement of knockdown. Firefly luciferase was selected as the target protein for knockdown as both the mRNA and protein have short half-lives. This would avoid complications that might arise when testing a gene with a more stable mRNA or protein.
The optimization parameters tested in this study were plating cell density, ratio of transfection reagent to plasmid, relative amounts of different plasmids, amounts of complex added, and time post transfection for measurement of knockdown.
Materials and Methods
Three distinctly different cell types, all purchased directly from ATCC, were studied. We chose HeLa (ATCC® CCL-2™) to represent an easy to transfect, commonly used cell line. HeLa cells were plated at 10,000 and 20,000 cells per well (96-well plate) on the day prior to transfection. MCF7 (ATCC® HBT-22™), a breast adenocarinoma, is more difficult to transfect and was selected to represent cells with more differentiated functions. MCF7 cells were plated at 10,000, 15,000, or 20,000 cells per well (96-well plate) on the day prior to transfection. Our third test system used RAW 264.7 (ATCC® TIB-71™), a virus-transformed mouse macrophage cell line which is very difficult to transfect. These are much smaller cells and were plated at 25,000 and 50,000 cells per well (96-well plate) on the day prior to transfection
The pGL3-Control vector (Promega) expressing firefly luciferase was selected, as there are commercially available plasmids containing sequences to knock down this protein. The pRL-SV40 vector (Promega) expressing Renilla luciferase was used for normalization and as an internal control. To keep the total amount of plasmid DNA equivalent in cotransfection experiments, pXM-Lac Z expressing b-galactosidase was used when shRNA plasmids were not part of the complex.
InvivoGen plasmids for knockdown were used to generate short hairpin RNAs (shRNA) in the cells. The psiRNA-LucGL3 plasmid expresses siRNA targeting the LucGL3 gene. Transfection of siRNA or plasmids containing knockdown sequences can have deleterious effects even when the targeted gene is not in the cells. Thus, as a control to monitor effects resulting from the transfection itself, we used psiRNA-LacZ which targets the LacZ gene (which should not be in any of the cells we used). Both plasmids contained the human 7SK RNA pol III promoter.
Transfection of plasmids
The FuGENE® HD Transfection Reagent package insert protocol was followed, as well as the recommendations for optimization in 96-well plates :
1. Plate cells at two different densities in two 96-well plates for each ratio to be tested. Incubate cells overnight
2. Form complexes in 96-well plates (round bottom, tissue-culture-treated)
3. Remove cell plates from incubator and with multi-channel pipetter add varying amounts of complex (8 µl, 5 µl, and 2 µl complex per well) to cell plates. Immediately return plates to incubator
4. Incubate for 1–2 days prior to assay for reporter gene expression
Two different ratios of reagent to plasmid DNA were tested. The relative proportions of the different plasmids were varied as described in Table 1. The internal control plasmid pRL-SV40 and the pGL3-Control Vector were in all complexes. For knockdown, the psiRNA-LucGL3 plasmid was included in the complex. Two types of controls were used for comparisons; one contained the psiRNA-LacZ (as an shRNA control), and one contained pXM-LacZ (no shRNA produced).
Reporter gene assays
Standard kits were used to measure protein expression. A dual luciferase kit (Promega) was used to measure both firefly and Renilla luciferase expression as relative light units (RLU). A beta-galactosidase staining kit (Roche Applied Science) was used to stain the percentage of cells transfected by the LacZ gene.
Calculation for knockdown
Calculation of the percent knockdown (KD) was done by comparing the expression of firefly luciferase in the wells transfected with the firefly luciferase shRNA plasmid (psiRNA-LucGL3} with the firefly luciferase expression in cells transfected with the control irrelevant plasmid (psiRNA-LacZ) after normalization to Renilla luciferase expression to account for any difference in cell number and transfection efficacy between the wells. The standard formula was used:
Results and Discussion
After initial comparisons between co-transfection and sequencial transfection of the expression plasmids and the shRNA plasmids (data not shown), co-transfection was determined to be the preferable method in order to avoid artifacts that might be caused by sequential transfection, and to save one step in the experiment. Since cells that take up one plasmid are likely to take up all plasmids transfected at the same time, it was assumed that all transfected cells would contain the three plasmids, and that some cells would contain no plasmids. Once the initial ranges were set for gene knockdown, percent transfection was verified for the conditions used for this co-transfection. Several wells transfected with a plasmid containing a LacZ gene in place of the shRNAplasmids (Table 1) were stained for b-galactosidase activity; 85–95% of the HeLa cells and 45–55% of the MCF7 cells were transfected. These are within expected ranges for single plasmid transfection in these cell lines.
Initial experiments with FuGENE® HD Transfection Reagent and the plasmids and cells we were testing did not result in consistent knockdown when the shRNA plasmids were used in excess of the expression plasmid in our co-transfections (Figure 1). The co-transfections did not yield sufficient levels of firefly luciferase for reproducible measurements of knockdown, and some cytotoxicity was observed. Thus, our next experiments focused on varying the relative proportions of expression and shRNA plasmids. We determined that in our test system the expression plasmids had to be in excess of the shRNA plasmid. We also determined in preliminary experiments that optimal knockdown was achieved when either the amounts of pRL-SV40 and pGL3-Control vector were similar, or the pGL3-Control Vector was in excess (data not shown). Optimization experiments were done using more pGL3-Control Vector than pRL-SV40.
Multiple experiments were performed within the narrowed ranges for optimization on knockdown as described in the Materials and Methods section on each of the cell lines with the following parameters tested:
- Two plating cell densities
- Two ratios of transfection reagent:plasmid DNA
- Amount of firefly luciferase plasmid > Renilla luciferase plasmid
- Amount of firefly luciferase plasmid + Renilla luciferase plasmid > shRNA plasmid
- Three amounts of complex
Typical results from individual experiments are shown in Figures 2 and 3. HeLa cells (Figure 2a) responded differently than RAW 264.7 cells (Figure 2b) and MCF7 cells (data not shown). Similar levels of knockdown were found in RAW 264.7 cells and MCF7 cells at 24 hours and at 48 hours post co-transfection. In contrast, lower knockdown was observed at 24 hours post co-transfection than at 48 hours post co-transfection in HeLa cells. Higher knockdown was achieved in both cell types when less of the shRNA plasmid was used in the complex (31% versus 54% of total plasmid being comprised of psiRNA-LucGL3). The general conclusion from our studies was that at either the 5:2 or the 6:2 ratio of reagent:DNA good knockdown (Figure 3) was achieved when the amount of expression plasmids was in excess of the shRNA plasmid.
As shown in Figures 2 and 3, when less shRNA plasmid was used than in the initial experiments described in Figure 1, many conditions resulted in consistently high knockdown. Once these initial parameters were established, FuGENE® HD Transfection Reagent proved to be a robust reagent for delivery of shRNA plasmids. Under optimized conditions, excellent knockdown was demonstrated in all three cell lines. When designing experiments to knock down normal cellular genes, conditions should be tested with lower amounts of the shRNA plasmid either by adding much less complex than in our experiment, or by adding irrelevant DNA to the complex to lower the effective concentration of the shRNA plasmid.
If shRNA and expression plasmids containing different promoters are used, or the proteins and cells targeted are different, optimization experiments may show that other ratios and amounts provide higher knockdown than was found in our test system.
FuGENE® HD Transfection Reagent efficiently transfects plasmids for expression of proteins; in this series of experiments we have clearly shown that it can also be used to transfect plasmids containing sequences that express functionally active shRNA in the three cell lines we tested. The protocols for delivery of expression plasmids and shRNA plasmids are identical, but care must be taken to optimize the amount of shRNA plasmid added to the cells. It should be emphasized that in our experiments, less shRNA plasmid in the complex yielded higher knockdown in the three cell lines used in our model systems. Once the parameters are established, there is a broad range over which consistently high knockdown is obtained, making FuGENE® HD Transfection Reagent a reagent of choice for these experiments.
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This article was originally published in Biochemica 2/2008, pages 15-18. ©Springer Medizin Verlag 2008