Cell and molecular biology studies have been facilitated by transfection, a technology that can deliver material like DNA, RNA, and protein into cells. However, there are multiple reagents on the market, and a careful study must be performed in order to find a suitable technique for your particular cellular system. One of the most effective re-agents that was available on the market in the early 90’s was DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-tri-methylammonium chloride) . This lipid has the ability to form cationic liposomes that can incorporate material of interest, forming a vesicle that is introduced into the cell and then released . The method has been reported to be convenient, reproducible, and efficient for numerous cell lines. Several products that rely on this technology are now available on the market. A combination of this compound with adenovirus has been shown to reach 20% of transfection efficiency in HepG2 cells . Other transfection re-agents using other types of lipids are available to transfect eukaryotic cells. However, the non-natural source of the cationic lipid present in most of these reagents is non-biodegradable, and can cause toxicity to the transfected cell. Presently, several lipids from natural sources are included leading to enhancement of transfection efficiency .
FuGENE® HD Transfection Reagent is new. Many authors have reported its high efficiency on many cell lines [4-6] which are known to be difficult to transfect (e.g., insect cells, leukemic T-cells). Several parameters can influence transfection efficiency of FuGENE® HD Transfection Reagent. The ratio of reagent to DNA, the amount of transfection complex added to the cells, the confluency of cells, and the incubation time of complex with the cells are parameters that are recommended to be optimized for each cell type studied.
The human hepatoma cell line ATCC® HB-8065™ (HepG2) is often used in studies on the regulation of liver-specific gene expression. However, numerous limitations of transfection in this cell line have been reported using common chemical and electrophysical methods . We therefore tested the efficiency of FuGENE® HD Transfection Reagent in this cell line. We also evaluated the transfection efficiency of the reagent in chick embryo hepatocytes (CEH). This primary cell culture system has been used for several years as an excellent model to study hepatic lipogenesis .
Materials and Methods
Cell culture and density
The HepG2 cells were maintained in culture as recommended by ATCC except that in the MEM media, 4 mM of l-glutamine (Sigma-Aldrich) was added. In preparation for transfection, cells were brought to a subconfluent state and then trypsinized, after which 2 x 106 cells were plated in 35-mm dishes. After 3 days of incubation at 37ºC in a 5% CO2 environment, the cells reached 95% confluency and were ready for the transfection assay.
CEH cells were prepared as previously described  and plated in 35-mm dishes. Each plate contains 1.5-1.8 x 106 cells and 2 ml of Waymouth medium (Sigma-Aldrich). Cells were transfected the following day.
Transfection efficiency was measured using the b-galactosidase reporter gene under the control of the cytomeg-alovirus virus (CMV) promoter. The plasmid was purified using a cesium chloride gradient in order to obtain highly pure DNA.
Transfection and analysis of transfection efficiency
CEH and HepG2 cells were prepared as specified above. The FuGENE® HD Transfection Reagent-DNA complex was then formed in the appropriate medium in the absence of serum and antibiotic for 15 minutes at room temperature. The cells were incubated in the presence of complex for 24 hours prior to detection of the b–galactosidase. The procedure was optimized by using different transfection re-agent:DNA ratios, by using different volumes of transfection complex, and by varying the time of complex formation, cell confluency, and the ratio of the transfected plasmids (e.g., pBluescript/CMV-bGal). All tested conditions are indicated in Tables 1 and 2. Four commercially available lipid-based reagents have also been tested under different conditions, and their best efficiencies obtained are shown in Figure 1 and 2.
In order to visualize the number of cells transfected, cells were fixed for 10 minutes in PBS containing 3% formaldehyde. After several washes in PBS, transfected cells were revealed in 1.5 ml of a solution containing 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide 2 mM MgCl2, 5% DMSO, and 0.02 mg/µl of X-gal. The plates were then incubated overnight at 30°C and observed under an invert-ed microscope. Representative fields of our best tested experimental conditions are depicted in the Figures 1 and 2.
Results and Discussion
In order to establish an efficient and convenient method to transfect both CEH and HepG2 cells, we have tested sever-al transfection reagents, and compared these results with those of the standard transfection procedure previously used in our laboratory.
Concerning the primary cell line CEH, five different param-eters have been analyzed (Table 1). We first evaluated the change in the volume of the FuGENE® HD Transfection Reagent, keeping a constant amount of vector DNA during the transfection. Our data reveal that the ratio 5:2 was optimal for CEH. Furthermore, we noticed that the time spent for the formation of transfection complex was an important parameter, and we established 15 minutes as an optimum (Table 1). We also noted that when the amount of transfection complex added to the cells was doubled (from 100 µl to 200 µl) there was a significant increase in the efficiency of the transfection. Another optimization step was to verify the influence of the variation of the ratio between pBlueScript and pCMV-bgal. Surprisingly, our data show no qualitative difference in the transfection efficiency. It might therefore be reasonable to believe that a transfection using between 0.5 µg and 1.5 µg of pCMV-bgal, or another reporter gene under the control of a strong promoter, may be sufficient for the detection of the transgene. However, we have to note that CEH are primary cells prepared each week in our laboratory from chick livers, resulting in a significant variation in cell confluency, which is very difficult to maintain constant. However, with serial dilution of the cell suspension, we were able to obtain a constant range in the cell confluency. In conclusion, our results demonstrate a positive correlation between the confluency of the cells and the efficiency of the transfection using FuGENE® HD Transfection Reagent (Table 1). Figure 1a shows a representative field of the optimal transfection efficiency obtained in CEH cells using FuGENE® HD Transfection Reagent. For comparison, the best result obtained with another commercially available transfection reagent is shown in Figure 1b.
As described in Table 2, three different parameters were studied with FuGENE® HD Transfection Reagent using the HepG2 cell line. In each case, two parameters remain con-stant, permitting us to compare the data in a qualitative way. First, the ratio between FuGENE® HD Transfection Reagent and the vector was tested, and our analysis reveals that using an 8:2 ratio appears to be optimal. As mentioned above for the CEH, the formation time of transfection complex is a relevant factor for the HepG2 cell line, as we noted a decrease of transfection efficiency when complex formation time was 25 minutes or more. Finally, when the amount of transfection complex was doubled, there was no qualitative difference between the efficiency, suggesting there is no need to use a higher quantity of transfection complex for the HepG2 cell line. As described for the CEH cells, several other commercial transfection reagents were also tested. Figure 2a shows a representative field of the optimal transfection efficiency obtained in HepG2 cells using FuGENE® HD Transfection Reagent (see conditions in figure legend). Figures 2b–2d show the best results obtained with other commercial transfection reagents.
Our optimization assay performed with both CEH and HepG2 cells showed that FuGENE® HD Transfection Reagent notably improved transfection efficiency compared with all other transfection reagents tested. The optimal transfection conditions for CEH primary cell line were to use cells cultured at about 80% confluency using 15 minutes of complex formation with a 5:2 FuGENE® HD Transfection Reagent:DNA ratio, and finally 100 µl of transfection complex added on the cells. For HepG2 cells, optimal conditions were 95% confluency for the cells, 15 minutes of complex formation with an 8:2 FuGENE® HD Transfection Reagent:DNA ratio, and 100 µl of transfection complex added on the cells. In conclusion, with a minimum of optimization, FuGENE® HD Transfection Reagent gave us superior transfection efficiency over all other reagents tested.
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This article was originally published in Biochemica 1/2007, pages 13-15. ©Springer Medizin Verlag 2007