Transfection of Adherent HEK293-EBNA1 Cells in a Six-Well Plate with Branched PEI for Production of Recombinant Proteins.

INTRODUCTIONFast and efficient production of recombinant proteins (r-proteins) remains a major challenge for the academic and biopharmaceutical communities. Pure r-proteins are often required in large amounts (hundreds of milligrams to gram quantities) when being developed as biotherapeutics, or in smaller quantities (milligrams) for high-throughput screening campaigns and structural or functional studies. Mammalian cells are often preferred over prokaryotic systems when expressing cDNAs of mammalian origin due to their superior capability to conduct elaborate post-translational modifications. Large-scale transfection of mammalian cells is now establishing itself as a "must-have" technology in the scientific community, as it allows the production of milligram to gram quantities of r-proteins within a few days after cDNA cloning into the appropriate expression vector. Although calcium-mediated large-scale transfection is very effective, polyethylenimine (PEI) is much easier to use: It binds to and precipitates DNA efficiently and the resulting DNA-PEI complexes are suitable for efficient transfection of mammalian cells. In particular, the branched isoform of PEI works well for adherent cells, as it promotes their attachment to the plastic surface. It is thus very useful in experiments requiring multiple medium exchanges or washing steps following transfection. Also, when used in conjunction with six-well CellBIND plates, branched PEI can be used to adhere transfected cells when establishing stable cell lines. This protocol describes the steps needed for successful transfection of HEK293 cells adapted to serum-supplemented or serum-free medium in adherent culture using branched PEI.