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

  • Karen Berg
  • Vanessa Nicole Schäfer
  • Natalie Tschorn
  • Jörn StitzEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2070)


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.

Key words

Mammalian cells Stable transfection Suspension culture Cell selection Transposon vectors Plasmid DNA 293F cells PEI IRES 



We like to thank Dr. Reto Eggenschwiler and Prof. Dr. Tobias Cantz, Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Germany, for the critical discussion of the manuscript and Danka Bratic for expertise technical support. This work was supported by Grant EFRE-0500031 by the European Regional Development Fund (EFRE) of the European Union to J.S.


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Karen Berg
    • 1
    • 2
  • Vanessa Nicole Schäfer
    • 1
  • Natalie Tschorn
    • 1
  • Jörn Stitz
    • 1
    Email author
  1. 1.Pharmaceutical Biotechnology, Faculty of Applied Natural Sciences, STEPs InstituteTH Köln—University of Applied SciencesLeverkusenGermany
  2. 2.Research Group Translational Hepatology and Stem Cell Biology, Cluster of Excellence REBIRTHHannover Medical SchoolHannoverGermany

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