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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
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2070)

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 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 

Notes

Acknowledgments

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.

References

  1. 1.
    Wurm FM (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol 22(11):1393–1398CrossRefGoogle Scholar
  2. 2.
    Baldi L, Hacker DL, Adam M et al (2007) Recombinant protein production by large-scale transient gene expression in mammalian cells: state of the art and future perspectives. Biotechnol Lett 29(5):677–684CrossRefGoogle Scholar
  3. 3.
    Cervera L, Kamen AA (2018) Large-scale transient transfection of suspension mammalian cells for VLP production. Methods Mol Biol 1674:117–127CrossRefGoogle Scholar
  4. 4.
    Robinson DK, Memmert KW (1991) Kinetics of recombinant immunoglobulin production by mammalian cells in continuous culture. Biotechnol Bioeng 38(9):972–976CrossRefGoogle Scholar
  5. 5.
    Gutiérrez-Granados S, Cervera L, Kamen AA et al (2018) Advancements in mammalian cell transient gene expression (TGE) technology for accelerated production of biologics. Crit Rev Biotechnol 38(6):918–940CrossRefGoogle Scholar
  6. 6.
    Schlaeger EJ, Christensen K (1999) Transient gene expression in mammalian cells grown in serum-free suspension culture. Cytotechnology 30(1–3):71–83CrossRefGoogle Scholar
  7. 7.
    Pham PL, Perret S, Doan HC et al (2003) Large-scale transient transfection of serum-free suspension-growing HEK293 EBNA1 cells: peptone additives improve cell growth and transfection efficiency. Biotechnol Bioeng 84(3):332–342CrossRefGoogle Scholar
  8. 8.
    Gorman C, Bullock C (2000) Site-specific gene targeting for gene expression in eukaryotes. Curr Opin Biotechnol 11(5):455–460CrossRefGoogle Scholar
  9. 9.
    Jordan M, Wurm F (2004) Transfection of adherent and suspended cells by calcium phosphate. Methods 33(2):136–143CrossRefGoogle Scholar
  10. 10.
    Geisse S (2009) Reflections on more than 10 years of TGE approaches. Protein Expr Purif 64(2):99–107CrossRefGoogle Scholar
  11. 11.
    Boussif O, Lezoualc’h F, Zanta MA et al (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci U S A 92(16):7297–7301CrossRefGoogle Scholar
  12. 12.
    Ho SCL, Bardor M, Feng H et al (2012) IRES-mediated tricistronic vectors for enhancing generation of high monoclonal antibody expressing CHO cell lines. J Biotechnol 157(1):130–139CrossRefGoogle Scholar
  13. 13.
    Grabundzija I, Irgang M, Mátés L et al (2010) Comparative analysis of transposable element vector systems in human cells. Mol Ther 18(6):1200–1209CrossRefGoogle Scholar
  14. 14.
    Ammar I, Izsvák Z, Ivics Z (2012) The sleeping beauty transposon toolbox. Methods Mol Biol 859:229–240CrossRefGoogle Scholar
  15. 15.
    Mátés L, Chuah MKL, Belay E et al (2009) Molecular evolution of a novel hyperactive sleeping beauty transposase enables robust stable gene transfer in vertebrates. Nat Genet 41(6):753–761CrossRefGoogle Scholar
  16. 16.
    Yant SR, Meuse L, Chiu W et al (2000) Somatic integration and long-term transgene expression in normal and haemophilic mice using a DNA transposon system. Nat Genet 25(1):35–41CrossRefGoogle Scholar
  17. 17.
    Donello JE, Loeb JE, Hope TJ (1998) Woodchuck hepatitis virus contains a tripartite posttranscriptional regulatory element. J Virol 72(6):5085–5092PubMedPubMedCentralGoogle Scholar
  18. 18.
    Zufferey R, Donello JE, Trono D et al (1999) Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors. J Virol 73(4):2886–2892PubMedPubMedCentralGoogle Scholar
  19. 19.
    Ivics Z, Hackett PB, Plasterk RH et al (1997) Molecular reconstruction of sleeping beauty, a Tc1-like transposon from fish, and its transposition in human cells. Cell 91(4):501–510CrossRefGoogle Scholar
  20. 20.
    Cui Z, Geurts AM, Liu G et al (2002) Structure-function analysis of the inverted terminal repeats of the sleeping beauty transposon. J Mol Biol 318(5):1221–1235CrossRefGoogle Scholar
  21. 21.
    Tom R, Bisson L, Durocher Y (2008) Transfection of HEK293-EBNA1 cells in suspension with linear PEI for production of recombinant proteins. CSH Protoc 2008:pdb.prot4977PubMedGoogle Scholar
  22. 22.
    Spidel JL, Vaessen B, Chan YY et al (2016) Rapid high-throughput cloning and stable expression of antibodies in HEK293 cells. J Immunol Methods 439:50–58CrossRefGoogle Scholar

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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