Comparazione di diversi markers di selezione per la generazione di pools di cellule stabili usando il sistema di trasposoni piggyBac

The development of stable high-producing mammalian cell lines represents a major bottleneck in the production of recombinant therapeutic proteins. Conventional transfection methods entirely rely on random transgene integration, a rare event which generally produces very heterogeneous cell pools requiring long screening procedures before the identification of clones with the desired characteristics in terms of productivity and stability. In this work, a transposon based gene delivery method was used for the generation of stable CHO DG44 pools expressing the tumor necrosis factor alpha receptor (TNFR)-human immunoglobulin G1 (IgG1) Fc fusion protein (TNFR:Fc). This system leverages the ability of the piggyBac (PB) transposase to catalyse the excision of a transgene flanked by specific PB termini from a donor plasmid and its integration into highly transcribed regions of the host genome. The selection of the clones was made throught three different antibiotics to test what is the best one and which is the best concentration. The cell pools with the highest productivity and lowest concentration of antibiotic were selected with 150 ug/mL of Zeocin.