Transient gene expression (TGE) in Chinese hamster ovary (CHO) cells with polyethylenimine (PEI) as a transfection reagent has been considered as an attractive method to produce recombinant proteins rapidly for pre-clinical studies. A high level of transfection efficiency, which is required for high-level TGE in CHO cells, can be achieved by increasing the PEI concentration. However, PEI induces cytotoxicity in a dose-dependent manner. To overcome this problem, Bcl-2 protein, an anti-apoptotic protein, was overexpressed in CHO cells (DG44). At a ratio of PEI to DNA (an N/P ratio) of 10, there were no significant differences in transfection efficiency and cell viability between Bcl-2 overexpressing and non-overexpressing cells. The transfection efficiency and cell viability were 2-11% and 83-92%, respectively. However, there were significant differences (P<0.05) in the transfection efficiency and cell viability between them at a higher N/P ratio. At an NIP ratio of 40, the transfection efficiency and cell viability of Bcl-2 non-overexpressing cells were 24-38% and 35-40%, respectively, while those of Bcl-2 overexpressing cells were 48-53% and 43-56%, respectively. Furthermore, compared with Bcl-2 non-overexpressing cells, more DNAs entered the Bcl-2 overexpressing cells, resulting in a higher rate of TGE per cell. PE-Annexin V apoptosis revealed that Bcl-2 overexpression suppressed PEI-induced apoptotic cell death at high N/P ratios. Taken together, Bcl-2 overexpression in CHO cells suppresses apoptotic cell death during PEI-mediated transient transfection, resulting in enhanced transfection efficiency and TGE. (C) 2013 Elsevier Ltd. All rights reserved.