Nucleofection is a highly effective gene transfer technique for human melanoma cell lines.
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 beta-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.
Han
Sandra Y
SY
Department of Dermatology, New York Harbor Healthcare System, New York University School of Medicine, New York, NY 10016, USA.
Gai
Weiming
W
Yancovitz
Molly
M
Osman
Iman
I
Di Como
Charles J
CJ
Polsky
David
D
D015153:Blotting, Western; D045744:Cell Line, Tumor; D002470:Cell Survival; D004247:DNA; D018274:Electroporation / Q000379:methods; D015870:Gene Expression; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008545:Melanoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D010957:Plasmids; D034741:RNA, Small Interfering; D014162:Transfection / Q000379:methods*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D008164:Luminescent Proteins; D034741:RNA, Small Interfering; D016159:Tumor Suppressor Protein p53; D004247:DNA; D001616:beta-Galactosidase