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

For the semi-quantitative assessment of mRNA capping efficiency, we took advantage of the -phage N protein-boxB RNA interaction, which normally regulates antitermination during transcription of -phage mRNAs (6) . The short N-terminal peptide of the N protein mediates its binding to the 17 nucleotides boxB RNA hairpins at nanomolar affinity (7) . The N peptide was fused the N-terminus of the NP868R African swine fever virus capping enzyme, resulting in a tethered capping enzyme (i.e. pCMV-N-NP868R), while four BoxBr hairpins were introduced to the 3 UTR of the Firefly Luciferase gene (i.e. pT710-Luciferase-4xBoxBr). The effects of this tethered capping system were tested on C3P3-G1 transcripts, together with various controls. HEK-293 cells were transfected as described above with the appropriate combination of plasmid using an empty dummy plasmid to transfect the same amount of DNA to all conditions. Luciferase reporter expression was monitored by conventional luciferin oxidation assays and normalized by hSEAP expression as described above.