Sustained high level transgene expression in mammalian cells mediated by the optimized transposon system.

Sustained, high level transgene expression in mammalian cells, especially stem cells, may be desired in many cases for studying gene functions. Traditionally, stable transgene expression has been accomplished by using retroviral or lentiviral vectors. However, such viral vector-mediated transgene expression is often at low levels and can be reduced over time due to low copy numbers and/or chromatin remodeling repression. The transposon has emerged as a promising non-viral vector system for efficient gene transfer into mammalian cells. Despite its inherent advantages over lentiviral and retroviral systems, system has not been widely used, at least in part due to the limited availability of vectors with manipulation flexibilities. Here, we seek to optimize -mediated transgene expression and generate a more efficient, user-friendly system. By engineering a panel of versatile vectors and constructing recombinant adenoviruses expressing transposase (PBase), we demonstrate that adenovirus-mediated PBase expression significantly enhances the integration efficiency and expression level of transgenes in mesenchymal stem cells and osteosarcoma cells, compared to that obtained from co-transfection of the CMV-PBase plasmid. We further determine the drug selection timeline to achieve optimal stable transgene expression. Moreover, we demonstrate that the transgene copy number of -mediated integration is approximately 10 times higher than that mediated by retroviral vectors. Using the engineered tandem expression vector, we show that three transgenes can be simultaneously expressed in a single vector with high efficiency. Thus, these results strongly suggest that the optimized system is a valuable tool for making stable cell lines with sustained, high transgene expression.