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

Autographivirinae is a family of bacteriophages encoding their own single-subunit RNAPs showing high specificity for their respective double-stranded promoters. The T7 DNA-dependent RNA polymerase (T7RNAP), their prototype, synthesizes RNA in vitro at a rate of 250 nucleotides/second and produces transcripts devoid of 5caps (22) . Given the importance of capping for mRNA stability and translation initiation (23) , we sought to determine if the translational efficiency of T7-transcripts by mammalian cells could be increased by mRNA capping. To investigate protein expression from T7 promoter-driven transcripts, the wild-type T7RNAP open reading frame (ORF) Figure 1 . The Firefly Luciferase gene reporter assay used for the optimization of the C3P3-G1 system. A diagram depicts the steps of gene expression from the C3P3-G1 expression system, using Firefly Luciferase as expression gene reporter. C3P3-G1 enzyme and T7 promoter-Luciferase plasmids (pT710-Luciferase) are co-transfected into HEK-293 or CHO-K1 cultured cells. C3P3-G1 mRNA are expressed by the RNA polymerase IIdependent IE1 human CMV promoter/enhancer. The transcripts are subsequently translated into C3P3-G1 protein, which accumulates in the cell cytoplasm where it mediates transcription and capping of luciferase mR-NAs from the pT710-Luciferase plasmid, while polyadenylation is encoded in a poly[A]-track from the pT710-Luciferase plasmid. Ultimately, luciferase protein is produced by mRNA translation, which is monitored by luciferin oxidation assay.