Efficient gene transfer into the human natural killer cell line, NKL, using the Amaxa nucleofection system.
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.
Maasho
Kerima
K
Receptor Cell Biology Section, Laboratory of Allergic Diseases, NIAID, NIH, 12441 Parklawn Drive, Room 205, Rockville, MD 20852, USA.
Marusina
Alina
A
Reynolds
Nicole M
NM
Coligan
John E
JE
Borrego
Francisco
F
D001426:Bacterial Proteins / Q000235:genetics; D002460:Cell Line; D018274:Electroporation; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D006801:Humans; D007694:Killer Cells, Natural / Q000502:physiology*; D008055:Lipids; D008164:Luminescent Proteins / Q000235:genetics; D018613:Microscopy, Confocal; D014162:Transfection / Q000379:methods*; D020691:rab GTP-Binding Proteins / Q000235:genetics; D018909:src Homology Domains / Q000235:genetics
D001426:Bacterial Proteins; D008055:Lipids; C086724:Lipofectamine; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D020691:rab GTP-Binding Proteins