In the following experiments transfection of human natural killer cell line NKL was examined:
Nucleofection with a vector containing yellow fluorescent protein (YFP) led to 48% YFP expressing cells in combination with high cell viability. In comparison, conventional electroporation resulted in an efficiency of only 8 %, lipid-mediated transfection did not show any detectable YFP expression.
Nucleofection parameters: Kit V + O-17
4 members of the Rab family, small GTPases involved in vesicular traffic, were expressed as YFP and GFP fusion proteins using the nucleofection technique. Characteristic localizations were not affected, whereas efficiency and optimal expression time were construct dependent.
Nucleofection with a YFP fusion of protein tyrosine phosphatase (SHP1) or a YFP fusion of SHP1's two SH2 domains showed 60% efficiency.
Natural killer (NK) cell lines are useful for studying facets of NK cell biology. Such cell lines are notoriously difficult to transfect by traditional methods, a fact that has hampered NK cell biology studies for a long time. To overcome this, we investigated the use of the Amaxa nucleofection system that directly transfers DNA into the nucleus of the cell. This technology has revolutionized transfection studies with heretofore relatively transfection resistant cell types such as T cells, B cells and dendritic cells. Despite these advances, NK cells and NK cell lines have remained relatively resistant to transfection, including nucleofection. In this study we employed cDNA for SHP1 and various Rab proteins cloned in enhanced green/yellow fluorescent protein (EGFP/EYFP) expression plasmids for transient transfections into NKL cells. The expression of EGFP/EYFP fusion proteins was analyzed by flow cytometry, immunoblot and confocal microscopic analyses. We achieved 40-70% transfection efficiency with high levels of expression in this cell line with 85-90% viability. The method used in this report proves to be far superior to existing methods for delivering DNA into this well studied NK cell line and, consequently, provides new experimental opportunities.