Advanced Establishment of Stable Recombinant Human Suspension Cell Lines Using Genotype-Phenotype Coupling Transposon Vectors.

Abstract

Stable mammalian, namely human, suspension cell lines play a pivotal role in red biotechnology production scenarios for the generation of state-of-the-art biologics. However, selection of genetically modified and highly productive cell populations-prior to the establishment of clonal lines-is often challenging. To overcome this limitation, we first describe an optimized transient transfection protocol using the inexpensive reagent polyethylenimine (PEI) and human 293F cells. Transposon donor vectors derived from Sleeping Beauty encompassing a cassette with the reporter gene encoding for the green fluorescent protein (GFP) coupled with an internal ribosome entry site (IRES) to the expression of puromycin-resistance are employed to readily detect transfected cells. Upon stable transfection in the presence and absence of transposase expression, respectively,and subsequent antibiotic selection, GFP expression using flow cytometry analysis, cell viability, and cell density can be examined over a range of up to 3 weeks. Owing to the integration of high vectorcopy numbers into the target cell genome, transposase-mediated transposition of transposon donor vectors is instrumental in the faster establishment of recombinant cell population as compared to the classical stable transfection of plasmid DNA.