C3P3-G1: first generation of a eukaryotic artificial cytoplasmic expression system

Jaïs, Philippe H; Decroly, Etienne; Jacquet, Eric; Le Boulch, Marine; Jaïs, Aurélien; Jean-Jean, Olivier; Eaton, Heather; Ponien, Prishila; Verdier, Fréderique; Canard, Bruno; Goncalves, Sergio; Chiron, Stéphane; Le Gall, Maude; Mayeux, Patrick; Shmulevitz, Maya

was subcloned downstream of the CMV promoter (pCMV-T7RNAP). When expressed in HEK-293 cells, T7RNAP was found in cytoplasm, whereas in-frame fusion of a SV40 nuclear localization signal at the amino-terminus redirected T7RNAP to the nucleus (Supplementary Figure S1 ) (24) . A reporter plasmid was generated by cloning the Firefly luciferase gene under control of the 10 T7-phage promoter from the major capsid protein (pT710-Luciferase) (25) . This reporter plasmid also contains a 40 adenosine track to ensure artificial polyadenylation, followed by the selfcleaving genomic hepatitis D ribozyme (26) . Following cotransfection of pCMV-T7RNAP and pT710-Luciferase plasmids in HEK-293 cells, luciferase expression was monitored by conventional luciferin oxidation assays and normalized ( Figure 1 ). As expected, low levels of luciferase expression were seen in transfected HEK-293 cells (Figure 2 ).