nucleofection is a highly effective gene transfertechnique for human melanoma cell lines

Nucleofection is a highly effective gene transfertechnique for human melanoma cell lines

Sandra Y. Han1*, Weiming Gai1*, Molly Yancovitz1, Iman Osman1, Charles J. Di Como2

and David Polsky1

1Department of Dermatology, New York Harbor Healthcare System, New York University School of Medicine, New York, NY, USA;2Aureon Laboratories, Inc., Yonkers, NY, USA

Correspondence: David Polsky, MD, PhD, Department of Dermatology, NYU School of Medicine, 522 First Avenue, Rm. 401, New York, NY

10016, USA, Tel.: +212 263 9087, Fax: +212 263 5819, e-mail: [email protected]

*Sandra Y. Han and Weiming Gai contributed equally to this study.

Sources of Support: Department of Veteran Affairs Medical Research Service.

Accepted for publication 13 December 2007

Abstract: Despite the increasing use of gene transfer strategies in

the study of cellular and molecular biology, melanoma cells have

remained difficult to transfect in a safe, efficient, and reproducible

manner. In the present study, we report the successful use of

nucleofector technology to transfect human melanoma cell lines.

This technology uses an empirically derived combination of cell

line-specific solutions and nucleofector programmes to

electroporate nucleic acid substrates directly into the cell nucleus.

Using a colorimetric b-galactosidase assay, we optimized

nucleofection parameters for 13 melanoma cell lines, leading to

maximum transfection efficiency and cell survival. The

combinations of cell solutions NHEM or T and nucleofector

programmes A-24 or U-20 produced the best results. We

compared nucleofection with two commercially available lipid-

based gene transfer systems, effectene and lipofectamine 2000

using a green fluorescent protein reporter vector. Nucleofection

demonstrated a 3- to 40-fold improvement in transfection

efficiency when compared with the lipid-based counterparts.

Nucleofection was also superior in transfecting small-interfering

RNA (siRNA) as determined by Western blot analysis. Lastly, we

applied nucleofection to the simultaneous transfection of a p53-

dependent luciferase plasmid and p53-siRNA. Experiments using

dual transfection showed knockdown of p53 expression and

silencing of the reporter plasmid. In conclusion, nucleofection is

highly effective for the transfer of nucleic acid substrates, singly or

in combination, into human melanoma cell lines.

Key words: co-transfection – gene transfer – melanoma –

nucleofection

Please cite this paper as: Nucleofection is a highly effective gene transfer technique for human melanoma cell lines. Experimental Dermatology 2008; 17:

405–411.

Introduction

Over the past decade, gene delivery systems have been

increasingly used to study and control gene expression.

Transfection of nucleic acid substrates has provided means

to upregulate gene expression, study transcriptional and

post-transcriptional regulation of various genes and gene

products, and downregulate expression of desired targets

(1). Non-viral approaches to gene transfer include those

mediated by chemical means, such as calcium phosphate,

DEAE dextran, and cationic lipo- or polysomes. Physical

techniques such as electroporation, hydroporation, ultra-

sound, and microinjection have also been used (1). Direct

injection, including the use of the ‘gene gun’, into whole

tissues such as muscle (1) and human skin has also been

performed (2) While some cells are easy to transfect,

melanoma cells, in particular, have remained difficult to

transfect with suitable efficiency. As an example, liposome-

mediated gene transfer yielded only 15% DNA delivery in a

murine melanoma cell line and 8% in a human melanoma

cell line. Moreover, the level of transgene expression in the

latter was undetectable (3). Despite the efforts to optimize

nucleic acid delivery, particularly with lipid-based delivery

systems or electroporation (4–8), these non-viral strategies

have not been widely successful in melanoma cells.

In contrast, viral vectors have been used successfully for

transfection of a wide variety of cell types, including mela-

noma. The use of lentivirus, in particular, resulted in high-

efficiency gene transduction in melanocytes and melanoma

cells and was an improvement over adenovirus- and retro-

viral-based vectors (9). An interesting recent report utilized

a retroviral vector which encoded the Cre recombinase to

DOI:10.1111/j.1600-0625.2007.00687.x

www.blackwellpublishing.com/EXDMethods

ª 2008 The Authors

Journal compilation ª 2008 Blackwell Munksgaard, Experimental Dermatology, 17, 405–411 405

remove an experimentally transfected SV-40 large T antigen

gene from melanocytes (10). This ‘reversible transfection’

of melanocytes with oncogenic SV-40 large T antigen

resulted in the short-term proliferation of melanocytes to

generate a large number of cells for treating experimentally

induced vitiligo. Once the cells were infected with the ret-

rovirus encoding the Cre recombinase, the SV-40 sequences

were excised from the melanocyte genomes, resulting in

non-transformed cells that could be transplanted into

experimental animals, and functioned to restore pigment to

the vitiliginous areas. While this is an exciting development

in melanocyte biology, the use of retroviruses generally suf-

fers from disadvantages such as the potential for insertional

mutagenesis, the time needed to construct vectors, and the

potential health and safety risks for laboratory personnel

(11–13). In addition, viral-based vectors are not suitable

for transient transfections. Studies of melanoma gene

expression have therefore been hampered by the lack of

effective and reproducible methods of gene transfer.

Nucleofection is a newer, non-viral gene delivery tech-

nology designed to expand upon the principles of electro-

poration for primary cells and hard-to-transfect cell lines.

Unlike traditional electroporation, nucleofection combines

cell-specific electrical parameters and Nucleofector Solu-

tion� (Amaxa AG, Cologne, Germany) to deliver genetic

material, including DNA, small-interfering RNA (siRNA),

and oligonucleotides directly to the nucleus. In doing so,

transfection is independent of cell division, leading to

increased efficiency. This method has already been success-

fully used in a number of cell types including keratinocytes

(14), human bone marrow-derived stem cells (15), endo-

thelial cells (16), glioblastoma cells (17), and melanocytes

(18). In this paper, we demonstrate that nucleofector tech-

nology can be successfully applied to many human mela-

noma cell lines for the efficient and simultaneous delivery

of DNA and siRNA, with results superior to that of com-

mercially available lipid-based transfection systems. More

efficient transfer of nucleic acid substrates into melanoma

cell lines may help to facilitate research efforts focusing on

the regulation of gene expression.

Methods

Cell cultureThirteen melanoma cell lines (SK-MEL 19, SK-MEL 23,

SK-MEL 29, SK-MEL 31, SK-MEL 85, SK-MEL 94,

SK-MEL 100, SK-MEL 103, SK-MEL 147, SK-MEL 173,

SK-MEL 187, SK-MEL 192, and SK-MEL 197) were a gift

of Dr Alan Houghton (Memorial Sloan-Kettering Cancer

Center, New York, NY, USA). The cells were maintained in

Dulbecco’s modified Eagle’s medium (DMEM; Cambrex,

East Rutherford, NJ, USA) supplemented with 10%

foetal calf serum (Gibco, Grand Island, NY, USA), 2.0 mM

l-glutamine (ATCC, Manassas, VA, USA), 50 U ⁄ ml peni-

cillin, and 50 lg ⁄ ml streptomycin. The cells were passaged

twice weekly for maintenance in logarithmic growth phase.

Plasmids and siRNAThe pRSV-lacZ plasmid encoding the Escherichia coli

b-galactosidase reporter gene was a kind gift of Dr Susan

Logan (New York University School of Medicine, New

York, NY, USA). Green fluorescent protein (GFP) was

expressed using the pmaxGFP plasmid (Amaxa, Cologne,

Germany), derived from the copepod Potellina sp. Plasmids

were amplified in the DH5a strain of E. coli and purified

using the Qiagen EndoFree Plasmid Maxi Kit (Qiagen,

Valencia, CA, USA).

The hdm2luc01 firefly luciferase reporter plasmid was a

kind gift of Dr Jeremy P. Blaydes (University of Southamp-

ton, UK) and has been previously described (19). The

plasmid contains the p53-responsive P2 promoter of the

HDM2 gene. The pRL-TK reporter plasmid (Promega,

Madison, WI, USA), which produces low-level, constitutive

expression of Renilla luciferase, was used as an internal

control.

The siRNA directed against human p53 was designed

and synthesized by Qiagen. The sequences of the siRNA

used are as follows: p53-siRNA-1: 5¢-GGA AAU UUG CGU

GUG GAG U-3¢ and 5¢-ACU CCA CAC GCA AAU UUC

C-3¢. Non-silencing siRNA was also purchased from Qia-

gen. The sequences of the control siRNA used were

5¢-UUC UCC GAA CGU GUC ACG U-3¢ and 5¢-ACG

UGA CAC GUU CGG AGA A-3¢.

NucleofectionCells were nucleofected using materials supplied in the

Amaxa Cell Line Optimization Nucleofector Kit�(Amaxa). For nucleofection of reporter plasmids, mela-

noma cells were grown to a confluence of 70–80%. Follow-

ing trypsinization for 10 min, 2 · 106 cells were suspended

in either 100 ll of Cell Line Nucleofector Solution T, R, or

V or melanocyte-specific NHEM� solution (Amaxa) in an

Amaxa-certified cuvette.

To determine optimal nucleofection conditions for mela-

noma cell lines, 2 lg of pRSV-lacZ was used as a reporter,

added into each cell suspension and pulsed with the pro-

grammes described in the manufacturer’s protocol for cell

line optimization. Each programme differs in the intensity

and length of electrical pulsation; and the combination of a

selected Nucleofector Solution and programme define the

optimal nucleofection parameters. For experiments to eval-

uate expression of GFP, 2 lg of pmaxGFP was mixed into

the cell suspension, and nucleofection was performed using

the optimal conditions already established for each cell line.

The negative control did not have a reporter plasmid added

to the cuvette prior to pulsation.

Han et al.

ª 2008 The Authors

406 Journal compilation ª 2008 Blackwell Munksgaard, Experimental Dermatology, 17, 405–411

For experiments to nucleofect siRNA, 2 · 106 SK-MEL

19 cells were suspended in Cell Line Nucleofector Solution

R and mixed with 10 lg of either nonsense ⁄ scrambled

siRNA or p53-siRNA-1. The final volume did not exceed

120 ll. The solution was then pulsed with the T-20 nucleo-

fector programme.

For experiments co-transfecting DNA and siRNA,

2 · 106 cells were mixed with 0.2 lg of hdm2luc01 and

0.01 lg of prL-TK in Cell Line Nucleofector Solution

NHEM or R, for SK-MEL 100 or 173 cells, respectively,

and 10 lg of either nonsense ⁄ scrambled siRNA or p53-siR-

NA-1. The final volume did not exceed 120 ll. The solu-

tion was then pulsed with the appropriate programme,

A-24 for SK-MEL 100 cells or T-20 for SK-MEL 173 cells.

Immediately following pulsation, 500 ll of pre-warmed

Roswell Park Memorial Institute (RPMI) 1640 (Sigma, St

Louis, MO, USA) was added to each cuvette. (RPMI media

was used in lieu of standard culture medium, as its lower

calcium concentration facilitates membrane recovery from

the nucleofection procedure.) The cells were transferred to

a 1.5-ml Eppendorf tube and incubated at 37�C. After

10 min, the nucleofected cells were transferred to a

24-well plate containing fresh, pre-warmed DMEM

(0.5 · 106 cells ⁄ well) and maintained at 37�C.

Lipid-based transfectionsThe SK-MEL 19, 173, and 197 cells were transfected using

effectene (Qiagen) or lipofectamine 2000 (Invitrogen,

Carlsbad, CA, USA). For transfection using effectene,

5 · 104 cells were plated onto a 24-well plate and grown

to a confluence of 50%. Transfection conditions were

optimized following the manufacturer’s specifications and

comprise the following: 0.2 lg of either pmaxGFP or

p53-siRNA transfected using a ratio of nucleic acid to lipo-

fection reagent of 1:50. For transfection with lipofectamine

2000, 0.75 · 105 cells were plated onto a 24-well plate and

grown to a confluence of 90%. Optimized conditions con-

sisted of 0.8 lg of either pmaxGFP or p53-siRNA transfect-

ed with a ratio of nucleic acid to lipofection reagent of 1:5.

All transfections were carried out in triplicate. All experi-

ments were performed at least twice.

Detection of p53Detection of p53 protein was accomplished using Western

blotting. Cells were lysed 48 h following transfection using

Laemmli’s sample buffer (BioRad, Hercules, CA, USA).

Fifty micrograms of cell lysate was fractionated using a

5–15% SDS–polyacrylamide gel (BioRad) and electropho-

retically transferred to a nitrocellulose membrane (What-

man, Brentford, Middlesex, UK). The membrane was

blocked with a solution of 8% non-fat milk in phosphate-

buffered saline (PBS) and 0.05% Tween-20 (Cambrex) for

2 h. The blocking solution was changed, and the

membrane incubated overnight, rocking at 4�C with the

primary antibody directed against human p53 (Ab-6,

1:1000; Oncogene, Cambridge, MA, USA). Following three

washes with a solution of PBS and 0.05% Tween-20, the

membrane was incubated, rocking at room temperature

for 1 h with the anti-mouse horseradish peroxidase

(HRP) secondary antibody (1:3000; Santa Cruz Biotech-

nology, Santa Cruz, CA, USA). Proteins were visualized

on an autoradiography film (LabScientific, Inc., Living-

ston, NJ, USA) using the SuperSignal West Pico chemilu-

minescent system (Pierce, Rockford, IL, USA). Equal

loading of lanes was verified using anti-Ran, (C-20, 1:100;

Santa Cruz Biotechnology) as a primary antibody with

anti-goat HRP secondary antibody (1:3000).

Detection of b-galactosidase activityEfficiency of pRSV-lacZ transfection was detected 24 h

post-nucleofection by measuring b-galactosidase activity

with the b-Gal Staining Kit (Invitrogen). The manufac-

turer’s protocol was followed for the assay. Cells staining

blue after 2 h were considered to be positive for b-galacto-

sidase expression. Cells were visualized at 100 · magnifica-

tion using an Olympus IX 70 microscope (Olympus

Corporation, Tokyo, Japan). Nucleofection efficiency was

calculated by taking the proportion of blue cells when com-

pared with the total number of cells in the field. Ten fields

were counted and the mean nucleofection efficiency calcu-

lated for each cell line. Cell survival after nucleofection

was estimated by comparing the number of viable cells

that underwent nucleofection with the number of control

cells plated at the same time, which did not undergo

nucleofection.

Detection of GFP expressionPlates were inspected for expression of GFP 24 h post-

transfection using a compound microscope equipped with

a Nikon Epi-Fluorescence Attachment (Nikon Eclipse

TS100; Nikon Corporation, Japan). Images were observed

and captured using both a 40 · and 100 · objective

(Nikon Digital Camera DXM1200F). Images of each sec-

tion were visualized using both light and fluorescence

microscopy with the same compound microscope. The

proportion of fluorescent cells to total cells was calculated

as the estimated transfection efficiency.

Detection of luciferase activityForty-eight hours following nucleofection, cells were har-

vested and luminescence from both the firefly and Renilla

reporters was determined with the dual luciferase reporter

system following the manufacturer’s instructions. Lumines-

cence measurements were taken with the Berthold Lumat

LB9507 luminometer (Berthold, Oak Ridge, TN, USA).

Relative luminescence units (RLU) are defined as the ratio

Nucleofection of melanoma cells

ª 2008 The Authors


between firefly and Renilla luciferase. All nucleofections

were performed in triplicate.

Results

To determine the optimal nucleofection conditions yielding

the greatest efficiency and the lowest cell mortality, cells

from three melanoma cell lines (SK-MEL 19, 103, and 173)

were initially tested. Each cell line was tested using each of

the four Nucleofector Solutions (R, T, V, and NHEM),

using various electrical parameters (‘programmes’) as rec-

ommended by the manufacturer. Nucleofection efficiency

was estimated using a colorimetric b-galactosidase assay.

Based on the results of initial experiments, an additional

round(s) of optimization was performed by testing various

programmes in combination with the solution that pro-

duced the best results in the initial experiments. The spe-

cific programmes tested were based on advice from the

manufacturer. For example, Table 1 shows the results of

the optimization for line SK-MEL 19. The results of the

initial experiments using programme T-20 with solutions

T, R, or V are shown in panel A of Table 1. As these

parameters did not produce satisfactory results, solution

NHEM-Neo was tried in the subsequent experiment (panel

B of Table 1), and programmes T-20, U-20, and A-24 were

used. The best result was obtained with the programme

U-20, so an additional round of optimization was per-

formed using solution NHEM-Neo and various pro-

grammes of the ‘U’ series (panel C of Table 1). For this

line, the best combination of efficiency and cell viability

was obtained with programmes U-20 and U-22. Similar

experiments were carried out for other cell lines. An

example of nucleofection of the pRSV-lacZ plasmid for cell

lines SK-MEL 19, 94, and 173 is shown in Fig. 1.

For the SK-MEL 19, 94, and 173 cell lines, the NHEM,

T, or R Nucleofector Solutions in combination with pro-

grammes U-20, T-20, or A-24 demonstrated the greatest

nucleofection efficiencies. These results established a guide-

line to evaluate the optimal conditions for a larger set of

melanoma cell lines. In total, nucleofection optimization

experiments were performed on all 13 lines available in the

laboratory, and were successful for all lines tested. The final

nucleofection conditions and efficiencies for each cell line

are summarized in Table 2. Nucleofection efficiencies ran-

ged from a low of 20% in SK-MEL 187 to a high of 90%

in SK-MEL 94. Cell viability following nucleofection was

acceptable with 50–80% of cells remaining viable 48 h fol-

lowing the procedure. Solutions NHEM and T were very

effective, with seven cell lines optimally nucleofected using

Solution T, five using Solution NHEM, and one using

Solution R. Two programmes – U-20 and A-24 – emerged

as the most effective for nucleofection, and these were the

optimal programmes in 12 of the 13 cell lines.

Table 1. Optimization of nucleofection conditions for SK-MEL 19

Cell line Solution Programme

Efficiency

(%)

Mortality

(%)

A

SK-MEL 19 T T-20 <5 30

R T-20 <3 30

V T-20 <3 30

B

SK-MEL 19 NHEM-Neo T-20 20–30 80

NHEM-Neo U-20 50–60 80

NHEM-Neo A-24 <10 50

C

SK-MEL 19 NHEM-Neo U-11 10 50

NHEM-Neo U-14 20–30 60

NHEM-Neo U-15 10–15 60

NHEM-Neo U-16 5–10 50

NHEM-Neo U-17 20–30 50

NHEM-Neo U-20 70–80 40–50

NHEM-Neo U-22 70–80 40–50

Figure 1. Nucleofection of melanoma cell lines using pRSV-lacZ.

SK-MEL 19, 94, and 173 cells were nucleofected with pRSV-lacZ and

analysed 24 h following transfection. Nucleofection of the reporter

plasmid was detected using b-galactosidase. Cells expressing the

plasmid stain blue. Magnification is at 100·.

Table 2. Summary of optimized nucleofection conditions for

various melanoma cell lines

Cell line

Cell

solution Programme

Efficiency

(%)

Mortality

(%)

SK-MEL 19 NHEM U-20 70–80 40–50

SK-MEL 29 NHEM U-20 30–40 50

SK-MEL 85 T A-24 >80 20

SK-MEL 94 NHEM U-20 80–90 40–50

SK-MEL 100 NHEM A-24 50 20

SK-MEL 103 T A-24 50 30

SK-MEL 147 T U-20 50–60 30

SK-MEL 173 R T-20 80 20–30

SK-MEL 187 T U-20 20–30 40

SK-MEL 192 T A-24 30–40 50

SK-MEL 197 NHEM U-20 >80 <5

SK-MEL 23 T U-20 40–60 30–40

SK-MEL 31 T A-24 40 20

Han et al.

ª 2008 The Authors


Transfection using nucleofection was compared with

lipid-based gene delivery systems. Cells from three mela-

noma cell lines, SK-MEL 19, 173, and 197 were transfected

with pmaxGFP using the optimized conditions for nucleo-

fection, effectene, or lipofectamine 2000. The results in

Fig. 2 demonstrate significantly greater fluorescence with

nucleofection in SK-MEL 19 and 197 cells when compared

with transfection with effectene and lipofectamine. Similar

results were obtained for SK-MEL 173 cells (data not

shown). The overall efficiency of gene transfer was esti-

mated to be 80% for nucleofection in all three cell lines. In

contrast, transfection with effectene resulted in estimated

efficiencies of 2% in SK-MEL 19 cells, 5% in SK-MEL 173

cells, and 1% in SK-MEL 197 cells. Transfection with lipo-

fectamine 2000 yielded similar results in SK-MEL 19 cells,

but results were improved for SK-MEL 173 and 197 cells,

with estimated efficiencies of 25% and 10%, respectively.

Mortality of SK-MEL 173 and 197 cells for nucleofection

remained low and were similar to those listed in Table 2.

Of note, mortality of SK-MEL 19 cells was greater using

nucleofection when compared with lipid-based transfection,

but the net number of transfected cells was substantially

greater than either lipid-based system.

A comparison of these techniques was also made for the

transfer of siRNA. SK-MEL 19 cells were transfected with

siRNA directed against p53 using optimized conditions for

nucleofection, effectene, or lipofectamine 2000. Control

cells were transfected with nonsense ⁄ scrambled siRNA.

Forty-eight hours after transfection, cells were lysed, and

detection of p53 protein was accomplished using Western

blotting (Fig. 3). In the right panel, a significant reduction

in p53 expression was observed in those cells subjected to

nucleofection when compared with control cells. Transfec-

tion with lipofectamine 2000 (middle panel) resulted in a

partial reduction of p53. Transfection with effectene (left

panel) failed to produce a detectable reduction in p53

levels.

One of the major advantages of nucleofector technology

is the ability to deliver DNA and siRNA to cell nuclei using

a single nucleofection condition. To test the co-transfection

capabilities of nucleofection, SK-MEL 173 and 100 cells

were nucleofected with p53-siRNA (or nonsense ⁄ scrambled

siRNA) plus plasmid DNA encoding the HDM2 promoter

driving a luciferase reporter gene. The protocol followed

was identical to those used for single agent nucleofection

(Table 2).

As seen in Fig. 4, the RLU for SK-MEL 173 cells

co-transfected with hdm2luc01 and nonsense ⁄ scrambled

p53-siRNA was 105.6 ± 9.9. When SK-MEL 173 cells were

co-transfected with p53-siRNA and hdm2luc01, however,

the RLU fell to 20.5 ± 2.3. Similar results were observed

for SK-MEL 100 cells. These results demonstrate the suc-

cessful knockdown of p53 through nucleofection of siRNA,

thereby suppressing the transcription of the p53-responsive

reporter plasmid. In addition, these findings show that

simultaneous transfection of plasmid DNA and siRNA are

readily accomplished using nucleofection.

Discussion

Despite great investigative advances afforded by the advent

of gene transfer technologies, these methods have not been

applied widely to the study of melanoma biology. Efforts

to conduct transgene studies of melanoma have largely

been hampered by the lack of efficient transfection meth-

ods, especially among non-viral vectors (9). In this paper,

we demonstrate that nucleofector technology is a highly

effective method for transfecting nucleic acid substrates

individually or in combination into human melanoma cell

lines.

(a)

SK-MEL 19 SK-MEL 197

(b)

(c)

Figure 2. Comparison of GFP expression 24 h following transfection

with nucleofector or lipid-based gene transfer systems. SK-MEL 19 and

197 cells were transfected using nucleofection (row a), lipofectamine

2000 (row b), or effectene (row c). Images of the right-hand panel for

each cell line were captured under fluorescence microscopy and show

cells in a representative field expressing GFP. Images of the left-hand

panel for each cell line were captured under simultaneous light and

fluorescence microscopy and demonstrate the total number of cells in

the field. Bright white cells are those expressing GFP. Magnification is

at 100·, except for SK-MEL 197 rows b and c, which are at 40·.

Figure 3. Western blot comparing p53 protein expression following

siRNA transfection using three techniques. SK-MEL 19 cells were

transfected with p53-siRNA using effectene, lipofectamine 2000, and

nucleofection. Transfection with nonsense ⁄ scrambled siRNA was used

as a negative control. Forty-eight hours following transfection, cells

were harvested for Western blotting for p53 expression. The protein

Ran was used to confirm equal protein loading of each lane. c. siRNA,

control siRNA.


ª 2008 The Authors


In order to obtain the best combination of transfection

efficiency and cell survival, the optimal nucleofection con-

ditions must be determined. A flow chart of the optimiza-

tion strategy we employed is shown in Fig. 5. In general,

optimization follows a stepwise process, in which the first

step is the determination of the appropriate nucleofector

solution; this is followed by testing various electrical

parameters until the optimal combination of transfection

efficiency and low mortality is determined. Whereas the

manufacturer Amaxa has optimized conditions for human

melanocyte cultures and for two human and two mouse

melanoma cell lines, we successfully transfected 13 different

human melanoma cell lines. Through our rounds of opti-

mization experiments, we found that the conditions that

yielded the best results in 12 of the cell lines were Nucleo-

fector Cell Line Solution NHEM or T in combination with

programme U-20 or A-24 (Table 2). These two solutions

and programmes may therefore serve as the basis for initial

experiments nucleofecting melanoma cell lines for those

investigators who are using the Amaxa nucleofector system

for the first time. These combinations, however, should not

be considered a substitute for conducting a full series of

optimization experiments following the manufacturer’s

protocol to determine optimal nucleofection conditions for

each cell line. This is highlighted by the finding that in the

SK-MEL 173 cell line, optimal nucleofection occurred with

a combination of Solution R and programme T-20, in con-

trast to the other 12 cell lines. Conditions for human mela-

nocyte cultures and other melanoma cell lines optimized by

Amaxa are listed in Table 3. We have also listed three

human melanoma lines in which nucleofection was used to

transfer siRNA directed against BRAF (20).

Figure 4. Results of co-transfection of siRNA and plasmid DNA as

measured by luciferase activity. SK-MEL 173 and 100 cells were

co-transfected with p53-siRNA and hdm2luc01 using nucleofector

technology. The presence of p53 is required for activation of the

luciferase reporter plasmid. Nucleofection of nonsense ⁄ scrambled siRNA

with hdm2luc01 served as the negative control. Co-transfection with

functional p53-siRNA resulted in a significant decrease in luciferase

activity for both cell lines. c. siRNA, control siRNA; RLU, relative

luminescence units.

Step 1 – Select the best Nucleofection SolutionTransfect the cell line of interest using program T-20 and each of the Nucleofector

solutions. The solution which yields the highest combination of transfection efficiencyand lowest mortality is used for the next step

Step 2 – Select the optimal Electrical Parameters Transfect the cell line using selected Nucleofector solution and each of the three

programs, U-20, T-20 and A-24. The condition which results in the highest combinationof transfection efficiency and lowest mortality is used for the next step.

Step 3 – Optimize the Conditions Perform additional transfections using the selected Nucleofector solution with severalprograms related to the program selected in Step 2. The goal is to further maximize

efficiency and minimize mortality. The company can help select the appropriateprograms to test.

Figure 5. Flow chart of nucleofection optimization strategy. The chart

outlines the general principles and steps taken to optimize the

transfection of cell lines using the nucleofection method.

Table 3. Summary of available nucleofector

conditions

Cell line Programme Solution Substrate

Efficiency

(%)

Viable

cells (%)

Analysis

method

Normal human

melanocytes1

U-24 NHEM 2.5 lg eGFP 55 ± 8 55–60 n ⁄ a

A-3751 X-001 V 2 lg maxGFP 72 ± 2 97 FACS

A-20581 X-001 C 2 lg maxGFP 81 ± 2 94 ± 1 FACS

B16-F01 P-031 R 2 lg maxGFP 84 ± 1 90 ± 1 FACS

B16-F101 P-020 V 2 lg maxGFP 91 ± 6 96 ± 1 FACS

1205Lu2 K-017 R 100 pmol siRNA

anti-BRAF

90 Western blot

C81612 K-017 R 100 pmol siRNA

anti-BRAF

90 Western blot

UACC9032 K-017 R 100 pmol siRNA

anti-BRAF

90 Western blot

1Optimized conditions by Amaxa.2From Sharma et al. (20).

Han et al.

ª 2008 The Authors


Nucleofection was more effective at gene transfer than

two commercially available lipid-based systems in experi-

ments transfecting plasmid DNA or siRNA. In terms of the

latter, suppression of gene function by mediating degrada-

tion of target mRNA has numerous research and therapeu-

tic implications. Recent studies in the field have focused on

using siRNA to silence oncogenes or other genes contribut-

ing to melanoma development or progression (21–23), but

given the difficulty in transfecting melanoma cells with

acceptable efficiencies, no standard procedure has emerged.

We found that nucleofection was a highly useful technique

to transfect DNA, siRNA, or both simultaneously in a sin-

gle step as demonstrated in Fig. 4. This versatility offers

significant opportunities to manipulate in vitro systems.

Another advantage of this technology is its ease of use.

Experimental procedures consisting of preparing the sam-

ples, pulsing the solution, and plating the newly nucleofect-

ed cells can be accomplished in less than 20 min.

Furthermore, because the nucleic acids are delivered

directly to the cell nucleus, cell division is not required for

substrate incorporation into the nucleus. This reduces the

delay between nucleofection and expression.

The limited drawbacks to using this system include

decreased cell survival in some lines when compared with

lipid-based gene delivery following nucleofection. However,

given the significantly higher transfection efficiency, the

increased mortality is not a major limitation in melanoma

cell lines. Another disadvantage is the high cost of this

technology in comparison to other gene transfer systems.

In conclusion, nucleofector technology enables the highly

efficient transfection of melanoma cells, which have tradi-

tionally been resistant to gene transfer using other non-

viral methods. With the ability to transfer DNA, siRNA, or

both simultaneously, this technology offers great promise

in aiding future investigative efforts in the field of

melanoma.

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ª 2008 The Authors


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