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

The subcellular localization of the wild-type T7RNAP was investigated by indirect immunofluorescence. Cells were plated on poly-L-lysine coated coverslips and cotransfected as described above with both pCMV-T7RNAP (encodes the wild-type ORF of T7RNAP under control of the CMV promoter) and pCMVScript-EGFP used as a cytoplasmic control. Cells were also transfected with a plasmid containing the T7RNAP ORF placed downstream to NLS from SV40 large T-antigen (i.e. pCMV-NLS-T7RNAP). Transfected cells were fixed in 4% paraformaldehyde in PBS for 20 min at room temperature. Cells were permeabilized and nonspecific binding was blocked by 30 min treatment in PBS with 5% goat serum, 0.1% Triton X-100 and 0.02% sodium azide. The coverslips were incubated overnight at 4 • C with the mouse monoclonal IgG The tagged C3P3-G1 enzyme was imaged by indirect immunofluorescence. CHO-K1 cells were transfected with the pCMV-FLAGx3-NP868R-(G 4 S) 2 -K1ERNAP(R551S), which encodes for the NP868R-(G 4 S) 2 -K1ERNAP(R551S) C3P3 enzyme fused in-frame to the C-terminus of the FLAGx3 tag (12) . CHO-K1 cells were then stained as above, using a primary polyclonal F7425 rabbit IgG anti-FLAGx3 antibody (1:250; Sigma) (13) followed by secondary Alexa Fluor 568-conjugated goat anti-rabbit (1:800; Life Technologies). Cells transfected with FLAG-RLuc plasmid and cells stained only with secondary antibodies were used as controls. Slides were imaged on a Leica SP8 confocal microscope equipped with the appropriate laser excitation filters under oil immersion (i.e. ×40 lens) with image magnification and processed with Leica LAS-AF and ImageJ analysis software.