QIAGEN

Transfection

Improved transfection efficiency of HT-1080, a fibrosarcoma cell line, using SuperFect Reagent
J.E.J. Rasko, R.J. Gottschalk, S.F. Jue, and A.D. Miller Program in Molecular Medicine, Fred Hutchinson Cancer Research Center, Seattle, WA, USA A fundamental requirement for investigating the biology of eukaryotic cells is an efficient means of gene delivery and expression. Although a number of transfection methods are available, some exhibit poor reproducibility, and many mammalian cell lines remain difficult to transfect. One cell line of interest to our laboratory is HT-1080, which was derived in 1972 from a fibrosarcoma in a 35-year-old male (1). SuperFect Transfection Reagent is a new reagent that uses a novel activated-dendrimer technology to produce high transfection efficiencies even with difficult-to-transfect cells (2, 3). We have tested this new reagent on HT1080 cells and compared the resulting transfection efficiencies with those obtained using other transfection reagents and techniques. In addition, we have assessed the cells by examining simultaneously the level of surface human placental alkaline phosphatase (hPAP) and the uptake of propidium iodide using flow immunocytometry. Transfection methods A clone of HT-1080 (ATCC-CCL-121) adherent cells, which are difficult to transfect, was used for all experiments.
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On the day prior to transfection, 1 x 105 HT-1080 cells were plated in 60-mm dishes (approximately 30% confluence) and incubated overnight at 37C and 5% CO2. Cells were transfected with the hPAP-encoding plasmid pLAPSN (4, 5). All transfection experiments were performed in triplicate and repeated at least once. Transfection using SuperFect Reagent was performed as described in the SuperFect Transfection Reagent Handbook and optimized as recommended. We examined combinations of 4, 10, or 20 l SuperFect Reagent with 2 g pLAPSN DNA; 5, 10, 25, or 50 l SuperFect Reagent with 5 g DNA; and 20, 50, or 100 l SuperFect Reagent with 10 g DNA in a total volume of 150 l DME medium. The mixture was incubated for 10 min at room temperature to allow transfection-complex formation. One ml DME medium containing 10% fetal bovine serum was added and the mixture was layered on phosphatebuffered saline (PBS)-rinsed HT-1080 cells. Cultures were incubated for 2, 4, 8, or 12 h, after which the dishes were rinsed with PBS. Medium was replenished and the cells were incubated for 48 h to allow for expression of the marker protein.

Transfection

QIAGEN

Electroporation of 2 x 106 HT-1080 cells was performed with 10 g pLAPSN DNA in a 0.4-cm cuvette. A capacitance of 960 F was used with actual voltages of 260 V and time constants between 17 and 18.5 msec. Optimized calcium phosphate coprecipitation of 5 x 105 cells per 60-mm dish was performed as previously described (6). Lipid-based transfection reagents were optimized and used according to the manufacturers instructions. Evaluation of transfection efficiencies Transient expression of surface hPAP was determined using flow cytometric evaluation of binding of a specific monoclonal antibody (Dako Denmark, clone 8B6) using a standard protocol (7). Binding was revealed by secondary staining using anti-mouse antibody conjugated to fluorescein isothiocyanate (FITC) or phycoerythin. Nonviable cells were detected by staining with propidium

iodide (1 g/ml). Between 10,000 and 20,000 cells were examined in a Becton-Dickinson FACScan device using CELLQuest software. Clumps and debris were excluded using forward and side-scatter windows. Stable expression of introduced DNA was determined following incubation of cells in the presence of G418 (750 g/ml) for 10 days (5). Stable expression was quantified by counting colonies stained using Coomassie Blue on single dishes. The plating efficiency was determined for each sample in parallel plates lacking G418 and the transfection efficiencies were corrected for clonogenic variation. Results The results of a typical FACS analysis of hPAP expression in HT-1080 cells transfected using SuperFect Reagent are shown in Figure 1. Using a two-parameter approach, we evaluated both transfection efficiency and effects on viability in the

A Propidium iodide fluorescence

hPAP-FITC fluorescence

hPAP-FITC fluorescence

Figure 1 FACS analysis of hPAP expression and viability in HT-1080 cells. s HT-1080 cells transfected with 5 g of A B pBluescript control DNA using 50 l SuperFect Reagent. s HT-1080 cells transfected for 2 h with 5 g pLAPSN DNA and 10 l SuperFect Reagent. Contour plots were scaled at 25% probability with one iteration of smoothing and a threshold of 1%.

Issue 2, 1997

QIAGEN

Transfection

Transfection efficiency
FACS Control pBluescript Control Electroporation Lipid L

Nonviable cells

References 1. Rasheed S., Nelson-Rees, W.A., Toth, E.M. Arnstein, P., and Gardner, M.B. (1974) Characterization of a newly derived human sarcoma cell line (HT-1080). Cancer 33, 10271033. 2. Haensler, J. and Szoka, F. (1993) Polyamidoamine cascade polymers mediate efficient transfection of cells in culture. Bioconjugate Chem. 4, 372379. 3. Tang, M.X., Redemann, C.T., and Szoka, Jr., F.C. (1996) In vitro gene delivery by degraded polyamidoamine dendrimers. Bioconjugate Chem. 7, 703714. 4. Halbert, C.L., Alexander, I.E., Wolgamot, G.M., and Miller, A.D. (1995) Adeno-associated virus vectors transduce primary cells much less efficiently than immortalized cells. J. Virol. 69, 14731479. 5. Miller, D.G., Edwards, R.H. and Miller, A.D. (1994) Cloning of the cellular receptor for amphotropic murine retroviruses reveals homology to that for gibbon ape leukemia virus. PNAS 91, 7882. 6. Miller, A.D., Miller, D.G., Garcia, J.V., and Lynch, C.M. (1993) Use of retroviral vectors for gene transfection and expression. Methods Enzymol. 217, 581599 7. Rasko, J.E.J., Metcalf, D., Gough. N.M., and Begley, C.G. (1995) The cytokine receptor repertoire specifies autocrine growth factor production in factor-dependent cells. Experimental Hematol. 23, 453460.

Lipid C SuperFect Reagent (25 l) SuperFect Reagent (10 l) SuperFect Reagent (5 l) 0 10 20 30 40 0 20 40 60

% Transfection efficiency

% Nonviable cells

Figure 2 Percentage transient expression levels and nonviable cells in HT-1080 cells transfected with pLAPSN DNA using different methods. Cells were assessed by flow cytometry 48 h after transfection. Cellular debris from lysed cells was excluded using forward and side-scatter windows. Negative controls for antibody staining and plasmid DNA (pBluescript DNA transfected into HT-1080 using SuperFect Reagent) were included in the analysis. The results show the mean value + standard deviation.

same cell population. Panel A shows HT-1080 cells transfected with 5 g of the control plasmid pBluescript using an excess (50 l) of SuperFect Reagent. This panel shows that even with an excess of SuperFect Reagent, 44% of cells were still viable (lower left region) while 56% of cells were nonviable (upper region). There was no expression of hPAP as shown by the empty lower right region. Panel B shows HT-1080 cells transfected with 5 g pLAPSN using 10 l SuperFect Reagent. The lower right region of Panel B shows that 21% of cells were transfected with pLAPSN and expressed hPAP, resulting in strong fluorescence. The upper region shows that 22% of the cells were nonviable. Results obtained using different transfection methods and conditions to introduce pLAPSN into HT-1080 cells are summarized in Figure 2. Three different volumes of SuperFect Reagent (5, 10, and 25 l) used for transfection of pLAPSN (5 g) resulted in high transfection efficiencies. SuperFect Reagent reproducibly delivered transient transfection efficiencies of greater than 20%. A
10

maximum efficiency of approximately 36% of the viable cells was obtained using 5 g DNA and 10 l SuperFect Reagent with an incubation time of 2 hours. These conditions are now used routinely in our laboratory. Extending the duration of exposure to SuperFect Reagent up to as much as 12 hours maintained transfection efficiencies above 23%. However, incubation times longer than 12 hours led to a further loss of approximately 20% of viable cells. In contrast, two commercially available lipid reagents (Lipid L and Lipd C in Figure 2) and electroporation produced transfection efficiencies of only 13%. As commonly found, transfection using calcium phosphate coprecipitation gave highly variable results in different experiments, with transfection efficiencies ranging from 5% to 29%. The right-hand panel of Figure 2 shows that the higher transient transfection efficiencies achieved with SuperFect Reagent did not correspond to a high level of cell death. Stable transfection efficiencies correlated with transient results.

Issue 2, 1997

Transfection

QIAGEN

1.25% stable transfection efficiency was achieved using 5 g pLAPSN and 10 l SuperFect Reagent and incubation for 2 hours. Longer incubation led to reduced efficiencies. Electroporation yielded 0.07% stable transfection efficiency. As with transient results, calcium phosphate co-precipitation yielded highly variable results for stable transfection, sometimes exceeding the levels obtained with SuperFect Reagent. Conclusions Our data show that with the difficult-totransfect cell line HT-1080, SuperFect Ordering Information
Product SuperFect Transfection Reagent (1.2 ml)

Transfection Reagent yielded up to 10-fold higher transfection efficiencies than other transfection reagents and techniques. In addition, the reproducibility we observed with SuperFect Reagent is unsurpassed. The shortened incubation time, low cytotoxicity and ease of use has made this an invaluable transfection reagent in our laboratory. s

Acknowledgements JEJR is supported by the Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation Fellowship, DRG-081.

Contents For 40 transfections in 60-mm dishes or 100 transfections in 12-well plates

Cat. No. 301305

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