With recent advances in molecular biology, it is now possible to construct genomic sequences and then evaluate the protein produced by those sequences. Ever-increasing portions of the genome are being investigated for their functions or for the effect sequence changes might have on them. Such study may entail testing the function of an unknown region of the genome, or mutating a sequence in a particular region to see how protein function or binding to other molecules might be affected. In these cases, using a complete plasmid for transfection is tedious, requiring time-consuming cloning, amplification, and purification of a plasmid construct. If multiple variants are to be tested, it would be much simpler if the PCR product could be tested directly, and a plasmid construct made only once the sequence of interest is identified.
We performed a series of experiments to determine if FuGENE® HD Transfection Reagent would deliver PCR fragments to cells and provide high expression of the transfected gene sequence. Transfection results of linearized as well as circular plasmids were set as baseline controls. PCR constructs were created using a commercially available kit. To test the concept we made the following investigations:
- What percentage of cells express the protein of interest?
- Are the levels of protein produced within a single cell sufficient for visual detection of transfected cells?
- Is the level of transfection comparable to that from a standard plasmid vector?
Materials and Methods
5,000 HeLa (ATCC® CCL-2™) cells per well were plated 1 day prior to transfection, using 96-well plates. This is a lower density than usually recommended for this cell line, and was used in order to observe expression in individual cells.
gWIZ-GFP (GeneTherapy Systems P 040400) and pGL3 Luciferase Reporter Vector (Promega E1741) were used for these experiments. gWIZ-GFP was linearized by Xho I restriction enzyme and purified.
FuGENE® HD Transfection Reagent and GenePORTER® transfection reagent (included in epiTAP Express® Kit, Gene Therapy Systems TAP 010220) were used according to the respective manufacturers’ instructions.
PCR products were prepared using the gene in both plasmids according to the instructions in the epiTAP Express® Kit (Figure 1).
Reagent-DNA complexes were formed using FuGENE® HD Transfection Reagent according to the package insert. Application Note 3 was used as a guideline for designing the plate layout . Since PCR constructs had never been previously assayed, we expanded the range of ratios tested. Different amounts of complex were added to the cells as recommended. All nucleic acids (circular plasmid, linearized plasmid, and PCR products) were prepared according to the same protocol in round-bottomed 96-well plates, although the ratios of reagent to DNA were different.
When the complex was formed using the PCR construct, only 25 µl of complex were prepared (instead of the normal 100 µl) to limit the amount of the PCR construct used. The epiTAP Express® Kit contains a transfection reagent called GenePORTER® which was tested according to the instructions in the kit.
1. Prepare diluted plasmid or PCR constructs at a final concentration of 20 µg/ml and 40 µg/ml (respectively) and vortex briefly to assure mixing. Calculate volume needed.
2. Add DNA to two columns of a new 96-well tissue culture-treated round-bottom plate.
3. Add 2-10 µl FuGENE® HD Transfection Reagent to the two columns of DNA wells to prepare different ratios (Table 1 lists the reagent:DNA ratios).
a. Insert pipette tips directly into the diluted plasmid, and gently mix the well contents by pipetting up and down 15 times.
b. When all complexes are formed, place the plate on an orbital shaker for 15-30 seconds to insure mixing. Incubate the plate at room temperature for 10-15 minutes.
4. Remove the cell plate from the incubator. Add different amounts of complexes (from Step 3) to the cell wells (7 µl, 5 µl, 3 µl, and 1 µl, as shown in Table 1).
a. Add complexes directly to the medium and mix gently so as to ensure adequate mixing without disturbing the cells.
b. Place the cell plate (with added complex) on an orbital shaker for 15-30 seconds for adequate mixing.
5. Return the cell plate to the incubator until observation and assay (24 or 48 hours).
Transfection of gWIZ-GFP circular and linearized plasmids
The standard circular plasmid resulted in higher levels of transfection than the linearized plasmid (Figure 2). Cells expressed varying amounts of protein; most HeLa cells showed some level of expression when transfected with the circular plasmid using FuGENE® HD Transfection Reagent. Complexes formed with the linearized plasmid resulted in low levels of expression; less than one third of the cells transfected with the linearized plasmid were visualized expressing GFP.
Transfection of gWIZ-GFP PCR product
As shown in Figure 3, when PCR product was used directly for transfection, sufficient GFP was produced within each cell to be observed in a fluorescent microscope. Even in this simple optimization experiment, conditions were found where approximately one third of the cells transfected with the PCR construct using FuGENE® HD Transfection Reagent expressed sufficient amounts of protein to be easily visualized. More transfected cells and more expression were observed with the PCR construct complex made using FuGENE® HD Transfection Reagent than with the GenePORTER® transfection reagent, which was supplied with the kit (data not shown). HeLa cells transfected with the FuGENE® HD Transfection Reagent complex morphologically looked similar to control cells.
Transfection of luciferase plasmid and PCR product
After visualizing transfection efficiency with the PCR construct via GFP expression, we quantified the difference in expression using luciferase (Figure 4). The pGL3 control vector was used as a template to amplify the luciferase gene, and a PCR construct was made using the epiTAP Express® System. A comparison between expression levels of luciferase 24 hours post transfection showed that the PCR construct, when transfected with FuGENE® HD Transfection Reagent, showed higher levels of expression than the pGL3 vector from which it was derived. Higher total expression was found in wells containing 10,000 cells per well than in wells plated with 5,000 cells per well. We also found that expression levels of luciferase were higher in the cells transfected with the PCR construct when FuGENE® HD Transfection Reagent was used than when the reagent supplied in the kit was used.
Discussion and Conclusions
We have shown that FuGENE® HD Transfection Reagent delivers circular plasmid, linearized plasmid, and PCR constructs containing only the gene of interest and regulatory sequences. Levels of expression in cells were sufficient such that GFP was readily observed in the fluorescent microscope following delivery of all three DNA sequences. Linearized plasmid is sometimes used to prepare stable transfectants, although it is generally agreed that expression levels with linearized plasmid are usually lower than with the circular plasmid from which it was obtained. We found, as expected, that circular plasmid resulted in the highest level of GFP expression; the linearized plasmid and the PCR construct resulted in similar levels of expression.
The GFP plasmid vector used in this experiment contains a very strong CMV promoter, and the lower expression with the PCR construct may have been a result of regulatory elements of the PCR construct rather than of construct delivery. This prediction is based upon the results with the luciferase plasmid, where a weaker promoter was compared with a luciferase PCR construct.
In the experiments done with the luciferase, FuGENE® HD Transfection Reagent was able to deliver sufficient levels of PCR construct derived from pGL3, such that luciferase expression in HeLa cells was higher than that from circular plasmid. This may be due in part to the pGL3 construct itself. We previously found that expression levels of pGL3 were about 10% of the expression level of another vector, pM1-Luciferase (data not shown). Further development of improved regulatory elements for the PCR construct should increase protein expression.
Transfection of PCR constructs directly into cells opens new avenues for exploring protein expression. Single or multiple base pair changes in the DNA sequence can be more rapidly tested at the protein level. FuGENE® HD Transfection Reagent is an excellent reagent for this application: levels of expression were comparable to those from conventional plasmid vectors, and were much higher than when the GenePORTER® transfection reagent was used for delivery of the PCR construct.
1. FuGENE® HD Transfection Reagent Application Note No. 3 / November 2006, “Protocol for optimizing transfection of adherent cell lines” Roche Cat. No. 04 960 629 001
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This article was originally published in Biochemica 4/2007, pages 22-25. ©Springer Medizin Verlag 2007