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DOI:10.1111/j.1600-0625.2007.00687.x www.blackwellpublishing.com/EXD

Methods

Nucleofection is a highly effective gene transfer technique for human melanoma cell lines Sandra Y. Han1*, Weiming Gai1*, Molly Yancovitz1, Iman Osman1, Charles J. Di Como2 and David Polsky1 1

Department of Dermatology, New York Harbor Healthcare System, New York University School of Medicine, New York, NY, USA; Aureon 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: david.polsky@med.nyu.edu *Sandra Y. Han and Weiming Gai contributed equally to this study. Sources of Support: Department of Veteran Affairs Medical Research Service. 2

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 lipidbased 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 p53dependent 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, ultrasound, 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, liposomemediated 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 melanoma. The use of lentivirus, in particular, resulted in highefficiency gene transduction in melanocytes and melanoma cells and was an improvement over adenovirus- and retroviral-based vectors (9). An interesting recent report utilized a retroviral vector which encoded the Cre recombinase to

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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 retrovirus 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 suffers 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 technology designed to expand upon the principles of electroporation for primary cells and hard-to-transfect cell lines. Unlike traditional electroporation, nucleofection combines cell-specific electrical parameters and Nucleofector Solution (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 successfully used in a number of cell types including keratinocytes (14), human bone marrow-derived stem cells (15), endothelial cells (16), glioblastoma cells (17), and melanocytes (18). In this paper, we demonstrate that nucleofector technology can be successfully applied to many human melanoma cell lines for the efficient and simultaneous delivery of DNA and siRNA, with results superior to that of commercially 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 culture Thirteen 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

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l-glutamine (ATCC, Manassas, VA, USA), 50 U ⁄ ml penicillin, and 50 lg ⁄ ml streptomycin. The cells were passaged twice weekly for maintenance in logarithmic growth phase.

Plasmids and siRNA The 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 Southampton, 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 Qiagen. 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¢.

Nucleofection Cells were nucleofected using materials supplied in the Amaxa Cell Line Optimization Nucleofector Kit (Amaxa). For nucleofection of reporter plasmids, melanoma cells were grown to a confluence of 70–80%. Following 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 melanoma cell lines, 2 lg of pRSV-lacZ was used as a reporter, added into each cell suspension and pulsed with the programmes 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 evaluate 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.

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Nucleofection of melanoma cells

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 nucleofector 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-siRNA-1. The final volume did not exceed 120 ll. The solution 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 37C. 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 37C.

Lipid-based transfections The 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 lipofection 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 consisted of 0.8 lg of either pmaxGFP or p53-siRNA transfected with a ratio of nucleic acid to lipofection reagent of 1:5. All transfections were carried out in triplicate. All experiments were performed at least twice.

Detection of p53 Detection 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 electrophoretically transferred to a nitrocellulose membrane (Whatman, Brentford, Middlesex, UK). The membrane was blocked with a solution of 8% non-fat milk in phosphatebuffered saline (PBS) and 0.05% Tween-20 (Cambrex) for 2 h. The blocking solution was changed, and the

membrane incubated overnight, rocking at 4C 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 Biotechnology, Santa Cruz, CA, USA). Proteins were visualized on an autoradiography film (LabScientific, Inc., Livingston, NJ, USA) using the SuperSignal West Pico chemiluminescent 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 activity Efficiency of pRSV-lacZ transfection was detected 24 h post-nucleofection by measuring b-galactosidase activity with the b-Gal Staining Kit (Invitrogen). The manufacturer’s protocol was followed for the assay. Cells staining blue after 2 h were considered to be positive for b-galactosidase expression. Cells were visualized at 100 · magnification using an Olympus IX 70 microscope (Olympus Corporation, Tokyo, Japan). Nucleofection efficiency was calculated by taking the proportion of blue cells when compared with the total number of cells in the field. Ten fields were counted and the mean nucleofection efficiency calculated 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 expression Plates were inspected for expression of GFP 24 h posttransfection 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 section 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 activity Forty-eight hours following nucleofection, cells were harvested and luminescence from both the firefly and Renilla reporters was determined with the dual luciferase reporter system following the manufacturer’s instructions. Luminescence measurements were taken with the Berthold Lumat LB9507 luminometer (Berthold, Oak Ridge, TN, USA). Relative luminescence units (RLU) are defined as the ratio

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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 recommended 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 produced the best results in the initial experiments. The specific 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 performed using solution NHEM-Neo and various programmes 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

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·.

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 programmes U-20, T-20, or A-24 demonstrated the greatest nucleofection efficiencies. These results established a guideline 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 ranged 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 following 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 R V

T-20 T-20 T-20

<5 <3 <3

30 30 30

SK-MEL 19

NHEM-Neo NHEM-Neo NHEM-Neo

T-20 U-20 A-24

20–30 50–60 <10

80 80 50

SK-MEL 19

NHEM-Neo NHEM-Neo NHEM-Neo NHEM-Neo NHEM-Neo NHEM-Neo NHEM-Neo

U-11 U-14 U-15 U-16 U-17 U-20 U-22

10 20–30 10–15 5–10 20–30 70–80 70–80

50 60 60 50 50 40–50 40–50

B

C

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Table 2. Summary of optimized nucleofection conditions for various melanoma cell lines

Cell line

Cell solution

Programme

Efficiency (%)

Mortality (%)

SK-MEL SK-MEL SK-MEL SK-MEL SK-MEL SK-MEL SK-MEL SK-MEL SK-MEL SK-MEL SK-MEL SK-MEL SK-MEL

NHEM NHEM T NHEM NHEM T T R T T NHEM T T

U-20 U-20 A-24 U-20 A-24 A-24 U-20 T-20 U-20 A-24 U-20 U-20 A-24

70–80 30–40 >80 80–90 50 50 50–60 80 20–30 30–40 >80 40–60 40

40–50 50 20 40–50 20 30 30 20–30 40 50 <5 30–40 20

19 29 85 94 100 103 147 173 187 192 197 23 31

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Nucleofection of melanoma cells

Transfection using nucleofection was compared with lipid-based gene delivery systems. Cells from three melanoma cell lines, SK-MEL 19, 173, and 197 were transfected with pmaxGFP using the optimized conditions for nucleofection, 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 estimated 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 lipofectamine 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. Transfection with lipofectamine 2000 (middle panel) resulted in a partial reduction of p53. Transfection with effectene (left

SK-MEL 19

SK-MEL 197

(a)

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.

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 successful 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.

(b)

Discussion (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·.

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 methods, 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.

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Step 1 – Select the best Nucleofection Solution Transfect the cell line of interest using program T-20 and each of the Nucleofector solutions. The solution which yields the highest combination of transfection efficiency and 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 combination of transfection efficiency and lowest mortality is used for the next step.

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.

In order to obtain the best combination of transfection efficiency and cell survival, the optimal nucleofection conditions must be determined. A flow chart of the optimization 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 optimization experiments, we found that the conditions that yielded the best results in 12 of the cell lines were Nucleofector Cell Line Solution NHEM or T in combination with

Step 3 – Optimize the Conditions Perform additional transfections using the selected Nucleofector solution with several programs related to the program selected in Step 2. The goal is to further maximize efficiency and minimize mortality. The company can help select the appropriate programs 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.

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 contrast to the other 12 cell lines. Conditions for human melanocyte 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).

Cell line

Programme Solution Substrate

Efficiency Viable Analysis (%) cells (%) method

Normal human melanocytes1 A-3751 A-20581 B16-F01 B16-F101 1205Lu2

U-24

NHEM

2.5 lg eGFP

55 ± 8

55–60

n⁄a

X-001 X-001 P-031 P-020 K-017

V C R V R

72 81 84 91 90

97 94 ± 1 90 ± 1 96 ± 1

FACS FACS FACS FACS Western blot

C81612

K-017

R

UACC9032

K-017

R

2 lg maxGFP 2 lg maxGFP 2 lg maxGFP 2 lg maxGFP 100 pmol siRNA anti-BRAF 100 pmol siRNA anti-BRAF 100 pmol siRNA anti-BRAF

1 2

± ± ± ±

2 2 1 6

90

Western blot

90

Western blot

Table 3. Summary of available nucleofector conditions

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

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Nucleofection of melanoma cells

Nucleofection was more effective at gene transfer than two commercially available lipid-based systems in experiments transfecting plasmid DNA or siRNA. In terms of the latter, suppression of gene function by mediating degradation of target mRNA has numerous research and therapeutic implications. Recent studies in the field have focused on using siRNA to silence oncogenes or other genes contributing 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 single 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 samples, pulsing the solution, and plating the newly nucleofected 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 traditionally been resistant to gene transfer using other nonviral 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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Nucleofection is a highly effective gene transfertechnique for human melanoma cell lines.