Ptk ren

The replicon was further modified by insertion of modified sequences in the nontranslated 3′ UTR as described in Table 1. Replicon plasmids were linearized using NotI, and RNA transcripts were synthesized in vitro using T7 RNA polymerase (MEGAscript T7; Thermo Fisher Scientific) for 4 h. pTK-Ren (Promega) expressing Renilla luciferase was cotransfected as a transfection control. pTK-Ren was linearized using XbaI. RNA transcripts were DNase treated (Thermo Fisher Scientific) and cleaned with a RNA Clean and Concentrator column (Qiagen). RNA integrity was confirmed on agarose gels, and the concentration was determined with Qubit fluorometric quantitation (Thermo Fisher Scientific).
Cells were seeded at 2 × 10⁴ cells per well in 96-well plates and transfected with 50 ng of replicon and 10 ng of pTK-Ren using the transfection reagent Lipofectamine 2000 (Thermo Fisher Scientific). At the 6 h posttransfection, cells were washed with phosphate-buffered saline (PBS) and harvested in passive lysis buffer (Promega), and the luciferase activities were measured using the dual luciferase reagent kit and GloMax multidetection system (Promega). The firefly luciferase readings were normalized to the Renilla value for each sample. In some formats investigating effects of gene KO, luciferase expression was further normalized to that of the parental WT A549 cell line control.

PS cells were seeded at 1.5 x10⁵ cells per well in 96-well plates (n = 3) and transfected with in vitro transcribed TBEV replicon RNA (100 ng/well) and 10 ng of XbaI linearized pTK-Ren (Promega) using Lipofectamine-2000 (Thermo Fisher Scientific). Cells were harvested in passive lysis buffer (Promega) at 12 to 72 hpt. Luciferase activity was measured using the Dual Luciferase reagent kit and GloMax multi detection system (Promega). pTK-Ren (Promega) expressing Renilla luciferase (RLuc), was included as an expression control. Quantitative data were obtained from three independent experiments.