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

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 vector copy 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.

Rapid establishment of stable retroviral packaging cells and recombinant susceptible target cell lines employing novel transposon vectors derived from Sleeping Beauty.

Viral vector particles derived from murine leukemia virus (MLV) mediate highly efficient stable gene transfer used in gene therapeutic approaches and in the generation of transgenic cell lines. However, the establishment of stable viral packaging cells (VPCs) is a time-consuming challenge. To overcome this limitation, we successfully generated novel Sleeping Beauty-derived transposon vectors entailing envelope and packaging expression cassettes as well as a transfer vector. Upon multiplexed transposition in human cells, VPC bulk populations yielding titers of over 1 × 106 transduction-competent vectors were established within three weeks. In contrast, conventional plasmid-based establishment of VPCs, conducted in parallel, took much longer and yielded significantly lower vector productivity and vector fitness. The generated MLV vectors decorated with the envelope proteins of ecotropic MLV PVC-211mc mediated efficient transduction of Chinese hamster ovary (CHO) cells. Cell susceptibility was further elevated upon recombinant expression of the murine ecotropic receptor mCAT employing a transposon vector.