Optimization of transient gene expression in mammalian cells and potentialfor scale-up using flow electroporation

The goals of this study were to identify mammalian cell Lines which could b e efficiently transiently-transfected and scaled-up for protein production. The transfection efficiencies of eight cell lines (NSO, NSO-TAg, CV-1, COS -7, CHO, CHO-TAg, HEK 293, and 293-EBNA) were measured using electroporatio n for DNA delivery and green fluorescent protein (Evans, 1996) as the repor ter gene. In addition, we have evaluated the effects of stable expression o f viral proteins, cell cycle manipulation, and butyrate post-treatment in s mall scale experiments. The cell lines varied widely in their GFP transfect ion efficiencies. Stable expression of simian virus 40 large T-antigen or E pstein Barr nuclear antigen failed to significantly increase transfection e fficiency above that seen in the parental lines. Aphidicolin (a DNA polymer ase inhibitor), which blocked cells from S or G2/M, brought about an increa se in transfection efficiency in two cell lines. The primary effect of buty rate (a histone deacetylase inhibitor) posttreatment was an increased inten sity of the fluorescent signal of green fluorescent protein, as measured by flow cytometry (1.0 to 4.2-fold, depending on the cell line). The combined use of aphidicolin pretreatment followed by butyrate treatment post-electr oporation yielded increases in fluorescence intensities ranging from 0.9 to 6.8-fold. Based on their high transfection efficiencies in small scale exp eriments, rapid growth, and ability to grow in suspension culture, CHO, CHO -TAg, and 293-EBNA were selected to assess the feasibility of using flow el ectroporation for large-scale transfections. Using secreted placental alkal ine phosphatase as a reporter, 293-EBNA cells produced the highest protein levels in both the presence and absence of butyrate. These data indicate th at flow electroporation provides an efficient method of DNA delivery into l arge numbers of cells for mammalian protein production.