Nucleofection is a highly effective gene transfer technique for human melanoma cell lines

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 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.

A brief overview of electroporation pulse strength–duration space: A region where additional intracellular effects are expected

“…Information regarding pulse strength and duration is more readily found in the patent literature than in research publications [65, 66, 67], which typically cite program protocols for pulsing designed by the manufacturer, and are not described in detail. In one general application, pulses are applied in pairs, with an initial large and relatively short pulse (2 – 10 kV/cm, 10 – 200 μ s) immediately followed by a smaller but longer pulse of 100 ms maximum duration [68].…”

Interaction of the Profilaggrin N-Terminal Domain with Loricrin in Human Cultured Keratinocytes and Epidermis

“…The culture of HaCaT cells was carried out as previously described (Yoneda et al, 2004). HaCaT cells were nucleofected using the Amaxa Cell Line Optimization Nucleofector Kit (Lonza, Walkersville, MD) (Han et al, 2008). For nucleofection, 10 mg of loricrin in the pcDNA3.1/V5-His vector or pFLAG467proF plasmid was used (Presland et al, 1997;Yoneda et al, 2010a).…”

Transdermal Gene Delivery by Functional Peptide-Conjugated Cationic Gold Nanoparticle Reverses the Progression and Metastasis of Cutaneous Melanoma

“…B16F10 is known as a hard-to-transfect murine tumor cell line. , However, from Figure A, transfection efficiency of AuPT in B16F10 reached highest of 1.71 × 10 7 RLU/mg total protein when the ratio of vector to pDNA is 1:8 (w/w). The zeta potential of the nanocomplexes with this ration was 16.81 ± 0.56 mV.…”