Drosophila S2 cells (Invitrogen) were cultured at 25°C in Schneider's Drosophila medium (GIBCO), supplemented with 10% heat inactivated foetal calf serum, 2mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. Firefly (Photinus pyralis) and Renilla reniformis luciferase sequences from the plasmids pGL3 and pRL-CMV (Promega) were cloned into pMT/V5-HisB (Invitrogen), generating pMT-Luc and pMT-Ren allowing Copper-inducible expression from a metallothein promoter.
Transfections were performed using Effectene transfection reagent (Qiagen) according to the manufacturer's recommendations. Luciferase expression was assayed using the Dual-Luciferase Reporter Assay System (Promega) and analyzed on a Tecan Ultra-evolution platereader. Double stranded RNA was generated by in vitro transcription from T7 promoter flanked PCR products. Viral stocks were prepared on low passage S2 cells and titered by end point dilution. Briefly, 25.000 S2 cells per well in a 96 well plate were inoculated with 10-fold dilutions of viral stocks. Cells were transferred to fresh medium at day 7 and CPE was monitored visually over 14 days. Viral titers were calculated according to the method of Reed and Muench.
Recombinant Sindbis virus expressing GFP during viral replication was generated by cloning enhanced GFP into the XbaI site of the double subgenomic Sindbis vector pTE3′2J (kindly provided by C. Rice)1 (link). The resulting plasmid was linearized and in vitro transcribed using the mMessage machine kit (Ambion). RNA was purified and electroporated into BHK-21 cells and supernatant was harvested and virus title determined by plaque assay on BHK cells.
Transfections were performed using Effectene transfection reagent (Qiagen) according to the manufacturer's recommendations. Luciferase expression was assayed using the Dual-Luciferase Reporter Assay System (Promega) and analyzed on a Tecan Ultra-evolution platereader. Double stranded RNA was generated by in vitro transcription from T7 promoter flanked PCR products. Viral stocks were prepared on low passage S2 cells and titered by end point dilution. Briefly, 25.000 S2 cells per well in a 96 well plate were inoculated with 10-fold dilutions of viral stocks. Cells were transferred to fresh medium at day 7 and CPE was monitored visually over 14 days. Viral titers were calculated according to the method of Reed and Muench.
Recombinant Sindbis virus expressing GFP during viral replication was generated by cloning enhanced GFP into the XbaI site of the double subgenomic Sindbis vector pTE3′2J (kindly provided by C. Rice)1 (link). The resulting plasmid was linearized and in vitro transcribed using the mMessage machine kit (Ambion). RNA was purified and electroporated into BHK-21 cells and supernatant was harvested and virus title determined by plaque assay on BHK cells.
