Transient Transfection in Mammalian Cells

Abstract

The aim of the presented work was to develop a cost-effective and easily scaleable transient transfection system with mammalian cells grown in serum-free suspension culture. For this purpose the cationic polyethylenimine (PEI) and the novel hybrid molecule dioleoyl-melittin (DOM) were used. Both substances are highly efficient transfection reagents for mammalian cells and have been described recently. The transfection complexes were made directly within the cell culture by consecutively adding plasmid and PEI (direct method). Alternatively, the DNA-PEI transfection complexes were performed in fresh medium (1/10 culture volume) and then added to the cells (indirect method).

Here we have optimized the PEI-mediated transfection conditions for HEK293 and 293EBNA cells grown in serum-free suspension culture for large scale experiments. In a second part of the investigation the efficiency of gene expression was studied in labscale as well as in fermentor scale by combining DOM with PEI to form transfection complexes.

Our data show the PEI-mediated transfection into EBNA cells can be used as a cost-effective transfection system in spinner culture scale up to 2-5L, to produce milligram amounts of recombinant protein. Fermentor scale experiments, however, are less efficient because the PEI- mediated transient gene expression is inhibited by the conditioned medium. Transfection experiments performed in the present of both DOM and PEI exhibited synergistic enhancement of gene expression in EBNA cells. The synergistic enhancement between DOM and PEI at low dose DNA (0, l-0,2μg/ml) was seen in spinner scale as well as in 10L and 23L fermentor