The PiggyBac transposon enhances the frequency of CHO stable cell line generation and yields recombinant lines with superior productivity and stability

Abstract

Generating stable, high-producing mammalian cell lines is a major bottleneck in the manufacture of recombinant therapeutic proteins. Conventional gene transfer methods for cell line generation rely on random plasmid integration, resulting in unpredictable and highly variable levels of transgene expression. As a consequence, a large number of stably transfected cells must be analyzed to recover a few high-producing clones. Here we present an alternative gene transfer method for cell line generation based on transgene integration mediated by the piggyBac (PB) transposon. Recombinant Chinese hamster ovary (CHO) cell lines expressing a tumor necrosis factor receptor:Fc fusion protein were generated either by PB transposition or by conventional transfection. Polyclonal populations and isolated clonal cell lines were characterized for the level and stability of transgene expression for up to 3 months in serum-free suspension culture. Pools of transposed cells produced up to fourfold more recombinant protein than did the pools generated by standard transfection. For clonal cell lines, the frequency of high-producers was greater following transposition as compared to standard transfection, and these clones had a higher volumetric productivity and a greater number of integrated transgenes than did those generated by standard transfection. In general, the volumetric productivity of the cell pools and individual cell lines generated by transposition was stable for up to 3 months in the absence of selection. Our results indicate that the PB transposon supports the generation of cell lines with high and stable transgene expression at an elevated frequency relative to conventional transfection. Thus, PB-mediated gene delivery is expected to reduce the extent of recombinant cell line screening.

Rapid recombinant protein production from piggyBac transposon-mediated stable CHO cell pools

“…Engineered transposable elements have been used to generate stably transfected CHO-and human embryonic kidney (HEK293)-derived cell lines for protein production (Alattia et al, 2013;Li et al, 2013;Matasci et al, 2011), and they have also served as gene transfer vectors for applications in gene therapy and gene discovery (Ding et al, 2005;Ivics et al, 1997;Mossine et al, 2013;Tsukiyama et al, 2011;Wu et al, 2006). piggyBac (PB), a class II transposable element originally derived from the cabbage looper moth, is one of the several transposons modified to function in mammalian cells (Fraser et al, 1996;Meir et al, 2011;Wilson et al, 2007).…”

Simple piggyBac transposon-based mammalian cell expression system for inducible protein production

“…Although use of the PBase for the generation of stably transfected mammalian cells for protein production holds much promise, the approach has seen only limited development and use to date (26). Reported here is a PB-based expression system for the generation of inducible, stably transfected mammalian cell cultures that has been in development and has been well tested in our laboratories during the past 3 years.…”

Mammalian Expression System and Improvisation for High Production

“…More than 500 mg/L in batch terminal shake flask was produced by using ACE platform cell line for monoclonal antibodies [4], [6].The cultivated mammalian cells can be integrated with the gene of interest by using the transposable elements like PiggyBac (PB) and sleeping beauty [7], [8]. In absence of selection pressure, the CHO cells transfected by PiggyBac showed stability for 3 month and an increase in the expression of fusion protein of tumor necrosis factor -Fc [9].…”