Optimisation of PEI-Mediated Transient Expression in Chinese Hamster Ovary Cells

The aim of this project is to develop transient gene delivery and expression strategies that can be employed in large-scale cultures of mammalian cells currently employed for therapeutic protein production (e.g. CHO, NSO). We have employed a model transfection system utilising the cationic polymer polyethylenimine (PEI) as a vehicle to deliver plasmid DNA encoding reporter gene constructs into suspension adapted Chinese Hamster Ovary cells. A key parameter in PEI-mediated transient gene expression is the PEI to DNA ratio. In our system expression was optimal at 10 motes PEI nitrogen to I mole DNA phosphate, which was conserved at different DNA to cell ratios. However, maximal reporter output was observed at a specific DNA:cell ratio. Addition of bovine serum albumin to growth medium was shown to increase both transgene expression (6 fold) and transfection efficiency (2 fold). Particle sizing data suggests that BSA acts to stabilise the PEI-DNA complexes at 300 nm, Therefore, these data imply that the specific properties of a PEI-DNA complex (particle size, charge etc), as well as the cellular "dose" of that complex may be important factors that regulate the uptake and intracellular fate of the recombinant DNA. In addition, small molecule inhibitors (SMI) were used to demonstrate that progression through the G2/M phase in the cell cycle is a pre-requisite for transgene expression. It was also observed that continued use of SMI throughout culture lead to an increase and an extension of transgene expression.