A Novel AIR-induced Gene Expression System in HEK.EBNA Cells and its Applications

Abstract

This work describes the first application of an efficient inducible gene expression system in HEK.EBNA cells. The transgene control system of choice is the novel acetaldehyde-inducible regulation (AIR) technology which has recently been shown to modulate transgene levels following exposure of cells to gaseous acetaldehyde (Weber et al., 2004). The AIR technology was engineered for gas-adjustable transgene expression in HEK.EBNA cells. AlcR transactivator and reporter plasmids containing a chimeric mammalian promotor (PAIR) were constructed for acetaldehyde-inducible expression of intracellular and secreted proteins. Several highly inducible transactivator encoding cell lines were established. Following transient transfection with reporter constructs and induction with acetaldehyde an up to 80-fold increase in transgene expression was achieved whereas leakiness of the system only accounted for 1-2% of the induction level. Thus, the AIR technology has been successfully applied to HEK.EBNA cells with tight repression and high induction levels. This system can be used for inducible expression of any desired recombinant protein. One application of the inducible gene expression system is controlled proliferation technology. An inverse correlation between growth rate and productivity of recombinant cell lines has been previously described (Fussenegger et al., 1998). Directing the metabolic capacity of HEK.EBNA cells towards production of recombinant proteins either by expression of cell cycle regulators or anti-apoptotic genes could be advantageous. This project comprises metabolic engineering of HEK.EBNA cells which upon expression of conditional cell cycle regulators uncouple the cell proliferation phase from the protein production phase. The p27 protein has been selected as a