High-yield production of human Dicer by transfection of human HEK293-EBNA1 cells grown in suspension

Bouvette, Jonathan; Korkut, Dursun Nizam; Fouillen, Aurélien; Amellah, Soumiya; Nanci, Antonio; Durocher, Yves; Omichinski, James G.; Legault, Pascale

To optimize transfection conditions, small-scale transfections were monitored over a 4-day period by withdrawing a 190-μL aliquot from the culture every 24 h to measure cell density, cell viability and recombinant Dicer expression. From this aliquot, a 10-μL volume was mixed with an equal volume of trypan blue and loaded on a hemocytometer slide to determine the cell density and viability using a Nikon Eclipse TS100 microscope. The remaining 180 μL of cell culture was pelleted, resuspended in 60 μL of lysis buffer (20 mM Tris pH 7.4, 50 mM NaCl, 10% Glycerol, 1 mM EDTA, 1 mM EGTA, 0.1% NP-40 and 0.5 mM TCEP [tris(2-carboxyethyl)phosphine]) and spun for 1 min at 16,000×g. The supernatant containing the cytoplasmic fraction was mixed with one volume of 2X Laemmli buffer and stored at − 20°C. All samples collected during transfection were heated at 95°C for 2 min, separated on a 7.5% SDS-PAGE and blotted on a nitrocellulose membrane (Bio-Rad) for Western-blot analysis. Dicer was detected using a mouse anti-His tag primary antibody (Medimabs #MM-0165-P), an HRP-conjugated anti-mouse secondary antibody (Cedarlane #115-035-044) and the Western Lightning Plus-ECL substrate (Perkin-Elmer #NEL103E001EA). A ChemiDoc MP (Bio-Rad) Imaging system was used for chemiluminescence detection, and band intensities were quantified using the ImageLab software version 5.2 (Bio-Rad).