Lorraine M. Albritton- Efficient transfection of fibroblast and epithelial cells with SuperFect Transfection Reagent

8/3/2019 Lorraine M. Albritton- Efficient transfection of fibroblast and epithelial cells with SuperFect Transfection Reagent

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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 DEAE-

dextranmediated transfections work well

on a very limited set of cell types and are

time-consuming to perform. Electroporationis rapid and efficient for a wide range

of cell types, but results in huge cell loss-

es and requires expensive equipment.

Although a number of simple-to-use lipid

reagents are now available which medi-

ate 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.

Materials and methods

NIH/3T3 cells (ATCC CRL-1658) and

293 cells (ATCC CRL-1573) were cul-

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 pGreen-

Lantern-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 lead-

ing liposome reagents, and optimized

according to the manufacturers instruc-

tions. Both DNASuperFect complexesand 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

Efficient transfection of fibroblast

and epithelial cells with SuperFectTransfection Reagent

Lorraine M. Albritton

Department of Microbiology and Immunology, University of Tennessee, Memphis,

Memphis, TN, USA

Ordering information forSuperFect TransfectionReagent can be found onpage 11.
http://www.qiagen.com/literature/qiagennews/0297/972impt.pdfhttp://www.qiagen.com/literature/qiagennews/0297/972impt.pdfhttp://www.qiagen.com/literature/qiagennews/0297/972impt.pdfhttp://www.qiagen.com/literature/qiagennews/0297/972impt.pdfhttp://www.qiagen.com/literature/qiagennews/0297/972impt.pdf


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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 (equiva-lent to 3 g per well of a 24-well plate).

DNASuperFect complexes yielded a

2-fold greater average number of trans-

fected 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 equivalentaverage 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 andthe 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 reproducibleexpression of transfected cDNA using

SuperFect ReagentAfter 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).

Comparison of transfection usingSuperFect 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 popula-

tions by fluorescent and phase-contrast

microscopy. Figure 2 demonstrates a

typical microscopic analysis of PA317

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

%o

fcellstransfected

0 10 20 30

60

40

20

0

40

Time post-transfection (h)

NIH/3T3 cells

Experiment 1

Experiment 2

Time post-transfection (h)

%o

fcellstransfected

0 10 20 30

60

40

20

0

40

293 cells


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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

liposome reagent. Similar results were

obtained with CRE cells.

Comparison of cytotoxicity

Transfection of NIH/3T3, CRE, and

PA317 cells using DNAliposome

Figure 2 Cells transfected

with pGL-1 usingSuperFect Reagent or lipo-somes. Living cultures areshown 48 h post-transfec-tion under fluorescentmicroscope and phase-contrast microscope.PA317 cells were trans-fected usingsA12.5 lSuperFect Reagent and2.5 g pGL-1, orsB10 lliposome reagent and2 g pGL-1. 293 cellswere transfected using

sC25 l SuperFectReagent and 5 g pGL-1,orsD25 l liposome

reagent and 5 g pGL-1.Transfections were per-formed in 35-mm disheswith cells at 75% conflu-ence. Magnification: 22x.

A B

C D

SuperFect Reagent Liposome Reagent

SuperFect Reagent Liposome Reagent

PA317 cells

293 cells


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complexes at all concentrations exam-

ined resulted in marked cell death with-in 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, trans-

fection 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 evenat 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 areformed and culture medium containing

serum is added before the complexes

are applied to the cells.

Comparison of the number of cellstransfected

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, DNASuperFectcomplexes 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 yieldedsignificantly better transfection results

than the widely used liposome reagent

that we tested. Transfections with

SuperFect Reagent resulted in several-

fold 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 approximately20 minutes preparation time. In addition,

SuperFect Reagent was significantly less

toxic to cells than the liposome reagent,

which caused marked cell death. s

References

1. Haensler, J. and Szoka,(1993) Polyamidoamine

cascade polymers mediate efficient transfection cells in culture.Bioconjugate Chem.4, 372379.

2. Tang, M.X., Redemann,C.T., and Szoka, Jr., F.C(1996) In vitro genedelivery by degradedpolyamidoamine den-drimers. BioconjugateChem. 7, 703714.

3. Chalfie, M., Tu Y.,Euskirchen, G., Ward,W.W., and Prasher, D.C

(1994) Green fluoresceprotein as a marker forgene expression. Scienc263, 802805.

4. Helm, R., Cubitt, A.B.,and Tsien R.Y. (1995)Improved green fluores-cence. Nature373,663664.

5. Zolotukhim, S., HauswirW.W., Guy, J., andMuzyczka, N. (1996) Ahumanized green fluorecent protein cDNAadapted for high level

expression in mammaliacells. Submitted.

SuperFect Transfection Reagent Liposome Reagent

Celldensity(cells/mm

2)

20 30 40 50

3000

2000

1000

60 70

Celldensity(cells/mm

2)

20 30 40 50

3000

2000

1000

60 70

Time post-transfection (h) Time post-transfection (h)

CRE cells PA317 cells

0 0

Figure 3 Cytotoxicity ofSuperFect Reagent andliposomes. CRE and

PA317 cells as indicatedwere transfected with2.5 g pGL-1 using12.5 l SuperFect Reagentor with 2 g pGL-1 using10 l liposome reagent.Transfections were per-formed in 35-mm disheswith cells at 75% conflu-ence. Cell densities(cells/mm2) were measuredusing phase-contrastmicroscopy at varioustimes post-transfection.

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Lorraine M. Albritton- Efficient transfection of fibroblast and epithelial cells with SuperFect Transfection Reagent