QIAGEN

Transfection

Efficient transfection of fibroblast and epithelial cells with SuperFect Transfection Reagent
Lorraine M. Albritton Department of Microbiology and Immunology, University of Tennessee, Memphis, Memphis, TN, USA Transient expression in mammalian cells is frequently used to establish the function of proteins encoded by newly isolated genes, as well as the effect of mutations on gene function. The choice of recipient mammalian cells has been limited to the few cell types that take up DNA efficiently with current transfection methods. Calcium phosphate and DEAEdextranmediated transfections work well on a very limited set of cell types and are time-consuming to perform. Electroporation is rapid and efficient for a wide range of cell types, but results in huge cell losses and requires expensive equipment. Although a number of simple-to-use lipid reagents are now available which mediate efficient transfection in a wider range of mammalian cells, the range is still limited. We investigated the efficiency of transfection with SuperFect Transfection Reagent*, a new non-lipid reagent based on activated-dendrimer technology (1, 2), in four adherent cell lines: human 293 fetal kidney epithelial cells, mouse NIH/3T3 fibroblasts, and the ecotropic and amphotropic retrovirus packaging cell lines, CRE and PA317, respectively.
* Ordering information for SuperFect Transfection Reagent can be found on page 11.

tured in DME medium supplemented with 8% donor calf serum (DCS). CRE cells (ATCC CRL-1858) and PA317 cells (ATCC CRL-9078) were cultured in DME medium supplemented with 8% fetal calf serum (FCS) (CRE and PA317 cells are derived from mouse NIH/3T3 fibroblasts). Cells were plated in 60-mm tissue culture dishes at 5060% confluence 2 days before transfection, and then seeded in 24-well culture plates or in 35-mm dishes at 4080% confluence 624 h prior to transfection. Transfections of plasmid pGreenLantern-1 (pGL-1), expressing a modified form of the green fluorescent protein (GFP) from Aequorea victoria (3, 4, 5), were performed using either SuperFect Reagent or one of the leading liposome reagents, and optimized according to the manufacturers instructions. Both DNASuperFect complexes and DNAliposome complexes were formed using the recommended optimal ratios of 5 l reagent per g plasmid DNA. The total amounts of DNA required for optimal transfection were determined on cells seeded at 4080% confluence. Transfections with SuperFect Reagent were performed using 12 g complexed DNA per well of a 24-well

Materials and methods NIH/3T3 cells (ATCC CRL-1658) and 293 cells (ATCC CRL-1573) were cul4

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plate and 2.55 g per 35-mm dish. In a separate experiment, 7.5 g complexed DNA was used per 35-mm dish (equivalent to 3 g per well of a 24-well plate). DNASuperFect complexes yielded a 2-fold greater average number of transfected cells 50 h post-transfection when 2 g rather than 1 g complexed DNA was used per well of a 24-well plate. Transfections with the liposome reagent were performed using 1 g complexed DNA per well of a 24-well plate and 2.55 g per 35-mm dish. DNA liposome complexes yielded equivalent average numbers of transfected cells 50 h post-transfection when 1 g, 1.5 g, or 2 g complexed DNA was used per well of a 24-well plate. DNASuperFect complexes were incubated with cells for 25 h, after which complexes were either removed prior to addition of medium containing 8% serum, or the medium was added without complex removal, and incubation was continued overnight. DNAliposome complexes were incubated with cells for 5 h, medium containing 16% serum was added, and incubation was continued overnight. Successful uptake of pGL-1 was determined by scoring green cells under a fluorescent microscope using an FITC (fluorescein isothiocyanate) filter set (for green fluorescent protein the excitation wavelength is 490 nm and the emission wavelength is 510 nm) on living cultures or on 10% formalin-fixed cells. The percentage of cells transfected was calculated as the number of green fluorescent cells (scored by fluorescent microscopy) over the total number of cells (scored by phase-contrast microscopy).

Rapid, extensive, and reproducible expression of transfected cDNA using SuperFect Reagent After transfection with SuperFect Reagent, the first few green fluorescent cells were visible under the fluorescent microscope within 8 h in all four cell types, indicating very rapid expression of the transfected pGL-1 sequences. Transfection efficiency increased with time, peaking at 4060% of 293 cells within 48 h post-transfection and at 1428% of NIH/3T3, CRE, and PA317 cells within 2436 h post-transfection. Transfection efficiencies with SuperFect Reagent were highly reproducible between independent experiments (Figure 1).
Experiment 1 Experiment 2

NIH/3T3 cells
% of cells transfected % of cells transfected 60 40 20 0 0 10 20 30 40 Time post-transfection (h) 60 40 20 0 0 10

293 cells

20

30

40

Time post-transfection (h)

Figure 1 Reproducibility with SuperFect Reagent. 293 and NIH/3T3 cells as indicated were transfected using 2.5 g pGL-1 and 12.5 l SuperFect Reagent. Transfections were performed in 35-mm dishes with cells at 75% confluence. Each graph shows transfection efficiencies obtained from two independent experiments.

Comparison of transfection using SuperFect Reagent and liposomes We compared transfection using SuperFect Reagent directly with transfection using one of the leading liposome reagents, and analyzed the effects on cell populations by fluorescent and phase-contrast microscopy. Figure 2 demonstrates a typical microscopic analysis of PA317
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A SuperFect Reagent

Transfection

B Liposome Reagent

PA317 cells

C SuperFect Reagent

D Liposome Reagent

293 cells
Figure 2 Cells transfected with pGL-1 using SuperFect Reagent or liposomes. Living cultures are shown 48 h post-transfection under fluorescent microscope and phasecontrast microscope. PA317 cells were transfected using s12.5 l A SuperFect Reagent and 2.5 g pGL-1, or s10 l B liposome reagent and 2 g pGL-1. 293 cells were transfected using C s 25 l SuperFect Reagent and 5 g pGL-1, or s 25 l liposome D reagent and 5 g pGL-1. Transfections were performed in 35-mm dishes with cells at 75% confluence. Magnification: 22x.

and 293 cells transfected with pGL-1 DNA. Cells transfected using SuperFect Reagent show less cell death (bottom panels) and a greater total number of transfected cells expressing GFP (top panels) than cells transfected using the
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liposome reagent. Similar results were obtained with CRE cells. Comparison of cytotoxicity Transfection of NIH/3T3, CRE, and PA317 cells using DNAliposome

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References 1. Haensler, J. and Szoka, F. (1993) Polyamidoamine cascade polymers mediate efficient transfection of cells in culture. Bioconjugate Chem. 4, 372379. 2. Tang, M.X., Redemann, C.T., and Szoka, Jr., F.C. (1996) In vitro gene delivery by degraded polyamidoamine dendrimers. Bioconjugate Chem. 7, 703714. 3. Chalfie, M., Tu Y., Euskirchen, G., Ward, W.W., and Prasher, D.C. (1994) Green fluorescent protein as a marker for gene expression. Science 263, 802805. 4. Helm, R., Cubitt, A.B., and Tsien R.Y. (1995) Improved green fluorescence. Nature 373, 663664. 5. Zolotukhim, S., Hauswirth, W.W., Guy, J., and Muzyczka, N. (1996) A humanized green fluorescent protein cDNA adapted for high level expression in mammalian cells. Submitted.

SuperFect Transfection Reagent

3000

Liposome Reagent

Cell density (cells/mm2)

2000

Cell density (cells/mm2)

CRE cells

3000

PA317 cells

2000

1000

1000

0 20 30 40 50 60 70 Time post-transfection (h)

0 20 30 40 50 60 70 Time post-transfection (h)

Figure 3 Cytotoxicity of SuperFect Reagent and liposomes. CRE and PA317 cells as indicated were transfected with 2.5 g pGL-1 using 12.5 l SuperFect Reagent or with 2 g pGL-1 using 10 l liposome reagent. Transfections were performed in 35-mm dishes with cells at 75% confluence. Cell densities (cells/mm2) were measured using phase-contrast microscopy at various times post-transfection.

complexes at all concentrations examined resulted in marked cell death within 2436 h post-transfection when cells were exposed at 4060% confluence (Figure 3). This cytotoxicity could be relieved on NIH/3T3, but not on CRE or PA317 cells, by using cultures higher in confluence (7580%). In contrast, transfection of NIH/3T3, CRE, and PA317 cells using up to 5 g DNASuperFect complexes per 35-mm dish (2 g per 24-well) did not result in cytotoxicity even at low cell densities. DNASuperFect complexes only exhibited marked cytotoxicity when used at 7.5 g per 35-mm dish. The difference in toxicity may in part be due to serum starvation of cells in the liposome procedure since DNA complexes are formed and exposed to cells in the absence of serum. In contrast, in the SuperFect protocol, DNA complexes are formed and culture medium containing serum is added before the complexes are applied to the cells. Comparison of the number of cells transfected We compared the average number of transfected cells per mm2 on identical cultures of cells exposed to the optimal amount of DNAreagent complexes.

Under optimal conditions for transfection with each reagent, DNASuperFect complexes yielded greater numbers of transfected cells than DNAliposome complexes: 6.9-fold greater with 293 cells; 2.6-fold greater with NIH/3T3 cells; and 3.3-fold greater with PA317 cells. Conclusions The new activated-dendrimerbased transfection reagent SuperFect yielded significantly better transfection results than the widely used liposome reagent that we tested. Transfections with SuperFect Reagent resulted in severalfold higher efficiencies, with a faster expression time, than parallel transfections with optimal amounts of the liposome reagent. Transfection-complex formation was equally rapid and simple with SuperFect Reagent as with the liposome reagent, both requiring approximately 20 minutes preparation time. In addition, SuperFect Reagent was significantly less toxic to cells than the liposome reagent, which caused marked cell death. s

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