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

To confirm that luciferase expression from NP868R-(G 4 S) 4 -T7RNAP depends on T7RNAP activity, transfected cells were exposed to ␣-amanitin, which inhibits polII but not phage RNAPs (29) . As expected, expression by NP868R-(G 4 S) 4 -T7RNAP was relatively resistant to ␣amanitin, in contrast to nuclear-based expression plasmid, which was effectively inhibited (Supplementary Figure S6) . 4 -K1ERNAP versus all other constructions: P < 0.05, Student's t-test. (E) Optimization of the polyadenylation/histone stem-loop region of DNA templates. A track of 40 adenosine residues of pK1E-Luciferase, which provides artificial polyadenylation to the transcripts was either removed or associated to the consensus stem-loop from human histone. This RNA element is involved in the regulation of stability and of translation efficiency in the cytoplasm and is functionally similar to a poly(A) tail in that it enhances translational efficiency and is dependent on mRNA capping. The corresponding pK1E-Luciferase with various sequence modifications were co-transfected with the pCMV-NP868R-(G 4 S) 2 To confirm the requirement of NP868R capping activity, the K282N mutation was introduced into the NP868R moiety of NP868R-(G 4 S) 4 -T7RNAP, which is predicted to suppress GTase activity by blocking NP868R-GMP adduct formation and thereby the transfer of GMP to 5 -diphosphate ends of mRNA (54) . As anticipated, the K282N mutation drastically impaired luciferase expression by C3P3-G1 (Supplementary Figure S7) .