Issue 2, 1997

Transfection

8

QIAGEN

Improved transfection efficiency of HT-1080, a fibrosarcoma cellline, 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 forinvestigating the biology of eukaryoticcells is an efficient means of gene deliveryand expression. Although a number oftransfection methods are available, someexhibit poor reproducibility, and manymammalian cell lines remain difficult totransfect. One cell line of interest to ourlaboratory is HT-1080, which wasderived in 1972 from a fibrosarcoma ina 35-year-old male (1).

SuperFect™ Transfection Reagent is a new reagent that uses a novelactivated-dendrimer technology to producehigh transfection efficiencies even with difficult-to-transfect cells (2, 3). Wehave tested this new reagent on HT-1080 cells and compared the resultingtransfection efficiencies with thoseobtained using other transfectionreagents and techniques. In addition,we have assessed the cells byexamining simultaneously the level ofsurface human placental alkalinephosphatase (hPAP) and the uptake of propidium iodide using flow immuno-cytometry.

Transfection methods

A clone of HT-1080 (ATCC-CCL-121)adherent cells, which are difficult totransfect, was used for all experiments.

On the day prior to transfection, 1 x 105

HT-1080 cells were plated in 60-mmdishes (approximately 30% confluence)and incubated overnight at 37°C and5% CO2. Cells were transfected withthe hPAP-encoding plasmid pLAPSN (4, 5). All transfection experiments wereperformed in triplicate and repeated atleast once.

Transfection using SuperFect Reagentwas performed as described in theSuperFect Transfection Reagent Hand-book and optimized as recommended.We examined combinations of 4, 10,or 20 µl SuperFect Reagent with 2 µgpLAPSN DNA; 5, 10, 25, or 50 µlSuperFect Reagent with 5 µg DNA; and20, 50, or 100 µl SuperFect Reagentwith 10 µg DNA in a total volume of150 µl DME medium. The mixture wasincubated for 10 min at room temperatureto allow transfection-complex formation.One ml DME medium containing 10%fetal bovine serum was added and themixture was layered on phosphate-buffered saline (PBS)-rinsed HT-1080cells. Cultures were incubated for 2, 4,8, or 12 h, after which the dishes wererinsed with PBS. Medium wasreplenished and the cells were incubatedfor 48 h to allow for expression of themarker protein.

1.007572 QNews 2/97 int. 24.09.1998 12:40 Uhr Seite 8

QIAGEN

Electroporation of 2 x 106 HT-1080cells was performed with 10 µg pLAPSNDNA in a 0.4-cm cuvette. A capacitanceof 960 µF was used with actual voltagesof 260 V and time constants between17 and 18.5 msec. Optimized calciumphosphate coprecipitation of 5 x 105

cells per 60-mm dish was performed aspreviously described (6). Lipid-basedtransfection reagents were optimized andused according to the manufacturers’instructions.

Evaluation of transfection efficiencies

Transient expression of surface hPAP wasdetermined using flow cytometricevaluation of binding of a specificmonoclonal antibody (Dako Denmark,clone 8B6) using a standard protocol(7). Binding was revealed by secondarystaining using anti-mouse antibodyconjugated to fluorescein isothiocyanate(FITC) or phycoerythin. Nonviable cellswere detected by staining with propidium

iodide (1 µg/ml). Between 10,000 and20,000 cells were examined in aBecton-Dickinson® FACScan™ deviceusing CELLQuest™ software. Clumpsand debris were excluded usingforward and side-scatter windows. Stableexpression of introduced DNA wasdetermined following incubation of cellsin the presence of G418 (750 µg/ml)for 10 days (5). Stable expression wasquantified by counting colonies stainedusing Coomassie® Blue on single dishes.The plating efficiency was determinedfor each sample in parallel plates lackingG418 and the transfection efficiencieswere corrected for clonogenic variation.

Results

The results of a typical FACS analysis ofhPAP expression in HT-1080 cellstransfected using SuperFect Reagent areshown in Figure 1. Using a two-parameterapproach, we evaluated both transfectionefficiency and effects on viability in the

Transfection

Issue 2, 19979

Figure 1 FACS analysis of hPAP expression and viability in HT-1080 cells. ■A HT-1080 cells transfected with 5 µg ofpBluescript control DNA using 50 µl SuperFect Reagent. ■B HT-1080 cells transfected for 2 h with 5 µg pLAPSN DNAand 10 µl SuperFect Reagent. Contour plots were scaled at 25% probability with one iteration of smoothing and athreshold of 1%.

Prop

idiu

m io

dide

fluo

resc

ence

hPAP-FITC fluorescence hPAP-FITC fluorescence

A B

1.007572 QNews 2/97 int. 24.09.1998 12:40 Uhr Seite 9

Issue 2, 1997

Transfection

10

QIAGEN

same cell population. Panel A showsHT-1080 cells transfected with 5 µg ofthe control plasmid pBluescript® usingan excess (50 µl) of SuperFect Reagent.This panel shows that even with anexcess of SuperFect Reagent, 44% ofcells were still viable (lower left region)while 56% of cells were nonviable(upper region). There was no expressionof hPAP as shown by the empty lowerright region. Panel B shows HT-1080 cellstransfected with 5 µg pLAPSN using 10 µl SuperFect Reagent. The lowerright region of Panel B shows that 21%of cells were transfected with pLAPSNand expressed hPAP, resulting in strongfluorescence. The upper region showsthat 22% of the cells were nonviable.

Results obtained using differenttransfection methods and conditions tointroduce pLAPSN into HT-1080 cellsare summarized in Figure 2. Threedifferent volumes of SuperFect Reagent(5, 10, and 25 µl) used for transfectionof pLAPSN (5 µg) resulted in high trans-fection efficiencies. SuperFect Reagentreproducibly delivered transient transfec-tion efficiencies of greater than 20%. A

maximum efficiency of approximately36% of the viable cells was obtainedusing 5 µg DNA and 10 µl SuperFectReagent with an incubation time of 2 hours. These conditions are now usedroutinely in our laboratory. Extendingthe duration of exposure to SuperFectReagent up to as much as 12 hoursmaintained transfection efficienciesabove 23%. However, incubation timeslonger than 12 hours led to a further lossof approximately 20% of viable cells.

In contrast, two commercially availablelipid reagents (Lipid L and Lipd C inFigure 2) and electroporation producedtransfection efficiencies of only 1–3%. Ascommonly found, transfection using calciumphosphate coprecipitation gave highlyvariable results in different experiments,with transfection efficiencies rangingfrom 5% to 29%. The right-hand panelof Figure 2 shows that the highertransient transfection efficiencies achievedwith SuperFect Reagent did not corre-spond to a high level of cell death.

Stable transfection efficienciescorrelated with transient results.

References

1. Rasheed S., Nelson-Rees,W.A., Toth, E.M. Arnstein,P., and Gardner, M.B.(1974) Characterizationof a newly derived humansarcoma cell line (HT-1080). Cancer 33,1027–1033.

2. Haensler, J. and Szoka, F.(1993) Polyamidoaminecascade polymers medi-ate efficient transfection ofcells in culture.Bioconjugate Chem. 4, 372–379.

3. Tang, M.X., Redemann,C.T., and Szoka, Jr., F.C.(1996) In vitro genedelivery by degradedpolyamidoamine den-drimers. BioconjugateChem. 7, 703–714.

4. Halbert, C.L., Alexander,I.E., Wolgamot, G.M.,and Miller, A.D. (1995)Adeno-associated virusvectors transduce primarycells much less efficientlythan immortalized cells. J. Virol. 69, 1473–1479.

5. Miller, D.G., Edwards,R.H. and Miller, A.D.(1994) Cloning of thecellular receptor foramphotropic murine retro-viruses reveals homologyto that for gibbon apeleukemia virus. PNAS 91, 78–82.

6. Miller, A.D., Miller, D.G.,Garcia, J.V., and Lynch,C.M. (1993) Use of retro-viral vectors for genetransfection and expression.Methods Enzymol. 217, 581–599

7. Rasko, J.E.J., Metcalf, D.,Gough. N.M., andBegley, C.G. (1995) Thecytokine receptor reper-toire specifies autocrinegrowth factor productionin factor-dependent cells.Experimental Hematol.23, 453–460.

Figure 2 Percentage transient expression levels and nonviable cells in HT-1080 cells transfected with pLAPSN DNAusing different methods. Cells were assessed by flow cytometry 48 h after transfection. Cellular debris from lysed cellswas 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 themean value + standard deviation.

Transfection efficiency

FACS Control

pBluescript Control

Electroporation

Lipid L

Lipid C

SuperFect Reagent (25 µl)

SuperFect Reagent (10 µl)

SuperFect Reagent (5 µl)

Nonviable cells

% Transfection efficiency

0 10 20 30 40

% Nonviable cells

0 20 40 60

1.007572 QNews 2/97 int. 24.09.1998 12:40 Uhr Seite 10

TransfectionQIAGEN

1.25% stable transfection efficiency wasachieved using 5 µg pLAPSN and 10 µlSuperFect Reagent and incubation for 2 hours. Longer incubation led toreduced efficiencies. Electroporationyielded 0.07% stable transfectionefficiency. As with transient results, calciumphosphate co-precipitation yielded highlyvariable results for stable transfection,sometimes exceeding the levelsobtained with SuperFect Reagent.

Conclusions

Our data show that with the difficult-to-transfect cell line HT-1080, SuperFect

Transfection Reagent yielded up to 10-fold higher transfection efficienciesthan other transfection reagents andtechniques. In addition, the reproducibilitywe observed with SuperFect Reagent isunsurpassed. The shortened incubationtime, low cytotoxicity and ease of usehas made this an invaluable transfectionreagent in our laboratory. ■

AcknowledgementsJEJR is supported by the Cancer Research Fund of theDamon Runyon-Walter Winchell Foundation Fellowship,DRG-081.

Ordering Information

Product Contents Cat. No.

SuperFect Transfection Reagent (1.2 ml) For 40 transfections in 60-mm dishes 301305or 100 transfections in 12-well plates

Why are these cells smiling?

They knowit’s ultrapure

QIAGEN DNA!

J QIAGEN Plasmid Kits — unsurpassed quality for more than a decade

J QIAfilter Plasmid Kits — the faster alternative

J EndoFree Plasmid Kit — endotoxin-free for ultimate plasmid DNA purity

Choose from 3 QIAGEN systems to suit your needs.

1.007572 QNews 2/97 int. 24.09.1998 12:41 Uhr Seite 11