Cai, Y., Bak, R.O., Krogh. L.B., Staunstrup, N.H., Moldt, B., Corydon, T.J., Schrøder, L.D. and Mikkelsen, J.G.
Notes: Researchers were looking for alternative methods to using transposase vectors carried by lentiviruses to insert genes into cellular DNA without the cytotoxicity that may occur if the transposase gene integrated into the genome. In this paper, the authors worked out a method to generate lentiviral particles that carried the transposase protein for delivery of genes at an equal efficiency as the conventional plasmid-based method. The reporter gene NanoLuc® luciferase was amplified from the pNL1.1[Nluc] Vector and cloned into a gag-pol-integrase-defective packaging construct. Firefly luciferase was cloned into the PB transposon lentiviral vector. Gag-pol constructs expressing the hyperactive piggyback (PB) transposase were also created. Lentiviral particles (LPs) were generated by cotransfection of several plasmids into 293T cells. One day prior to transduction, HeLa cells were seeded at a density of 104 cells/well in a 96-well plate, then NanoLuc® LPs with or without pseudotyping by Vesicular Stomatitis Virus envelope glycoprotein were added. After 48 hours, luminescence was measured using the Nano-Glo® Luciferase Assay System. To analyze how well the firefly luciferase gene was transferred, HaCaT and ARPE-19 cells were seeded at 1,000 cells/well in a 96-well plate one day before transduction with increasing amounts of either wildtype or mutated PBase/firefly luciferase transposon LPs. After ten days, the transduced cells were assessed for luminescence using the ONE-Glo™ Luciferase Assay System. HEK293 cells, primary keratinocytes and normal human dermal fibroblasts were seeded at 5,000 cells/well in 24-well plates the day before transduction and then incubated with either wildtype or mutated PBase/firefly luciferase transposon LPs. After eight days, firefly luminescence was measured. (4448)