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

Human erythropoietin in the culture media was assayed for effects on cell proliferation with the human UT7 leukemic cell line, which has megakaryocytic features and strictly depends on erythropoietin, GM-CSF, or IL3 for its growth (18) . UT7 cells were cultured as described elsewhere with slight modifications (19) in Alpha Modified Minimum Essential Medium Eagle supplemented with 10% fetal calf serum and 2 U/ml hEPO. Before each experiment performed in triplicate, cells were washed, and then serum-and growth factor-deprived by incubation for 24 h in Iscove's modified Dulbecco's medium carrying 0.4% bovine serum albumin. Then, serial two-fold dilutions (from 4 to 0.008 U/ml) of hEPO from the culture medium of transfected CHO-K1 cells or treated with recombinant hEPO (Epoetin ␤, Roche Laboratories) were added to the culture medium of UT-7cells. Cells were grown in 96-well plates for three days at 37 • C in 5% CO 2 atmosphere at 100% relative humidity. Cell proliferation was then assayed by addition of the Uptiblue reagent (Interchim, Montluçon, France), which is a viable cell counting reagent. Fluorescence intensity was read using a scanner (Typhoon, GE Healthcare), with excitation at 532 nm and emission at 580 nm. Human EPO released in culture medium was compared with the commercial Epoetin ␤ (Roche Pharmaceuticals, Basel, Switzerland). Differences of activity profiles between the commercial Epoetin ␤ and hEPO produced by CHO-K1 cells might be explained by the use of distinct methods for measuring hEPO or other differences related with the processing of Epoetin ␤ (20).