(5d) Enhancing Transient Gene Expression In CHOs Cells Through the Co-Transfection of the Product Gene and Bcl-Xl

Transient gene expression is gaining popularity as a method to rapidly produce recombinant proteins in mammalian cells.  Although significant improvements have been made, in terms of expression, more is needed for this method to compete with stable gene expression. Much improvement has come from transfection media optimization, transfection parameter optimization as well as altering culturing conditions to enhance productivity. Recent studies have also included using cell lines engineered for apoptosis resistance through the constitutive expression of an anti-apoptotic protein, Bcl-xL.  While significant improvement in yield has been observed there is still a significant time investment needed to generate the stable clones over-expressing Bcl-xL. In this study we examine an alternative method of using the benefits of anti-apoptotic gene expression to enhance the transient expression of bio-therapeutics, namely, through the co-transfection of bcl-xl and the product gene.  This method allows for fine tuning the transfection conditions for the optimal Bcl-xL expression level to both protect the cell from the harmful conditions of transfection and enhance specific productivity. For this study we have chosen the commercially relevant suspension CHO cell line together with the in-expensive and effective transfection reagent Polyethylenimine.  

We used a YFP vector to monitor transfection efficiencies and relative expression levels through the detection of YFP using flow cytometry and separately utilized a vector containing a gene for an Fc fusion protein to look at overall yield and apoptosis resistance.  Several conditions were tested and while there was a negligible effect on transfection efficiencies we found both a reduction in apoptosis and an increase in specific productivity for cultures co-transfected with the bcl-xL gene. Overall, by co-transfecting bcl-xL, minimal cost or complexity is added to a method of transient gene expression and yet significant improvements in product yields are achieved.