Strategies for Efficient Transfection of CHO-Cells with Plasmid DNA

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 801)

Abstract

Stable cell lines of Chinese hamster ovary (CHO) cells are the predominant source of commercial ­biopharmaceutical proteins. Because making suitable CHO cell lines is time-consuming and costly, ­preliminary experiments with transient expression are usually performed to optimize as many protein ­production parameters as possible. Here, we describe protocols for optimizing expression in transient expression experiments and isolating stable CHO cell lines using two types of self-made reagents, namely, lipoplexes and polyplexes.

Key words

Transfection Chinese hamster ovary Transient Stable Lipoplex Polyplex 

Notes

Acknowledgments

We thank Hannes Reisinger for technical help and Marion Tschernutter for her help with manuscript preparation.

References

  1. 1.
    Crystal, R. (1995) Transfer of genes to humans: early lessons and obstacles to success. Science 270, 404–410.PubMedCrossRefGoogle Scholar
  2. 2.
    Tripathy, S., Black, H., Goldwasser, E., and Leiden, J. (1996). Immune responses to transgene-encoded proteins limit the stability of gene expression after injection of replication-defective adenovirus vectors. Nat. Med. 2, 545–550.PubMedCrossRefGoogle Scholar
  3. 3.
    Sadelain, M. (2004) Insertional oncogenesis in gene therapy: how much of a risk? Gene Ther. 11, 569–573.PubMedCrossRefGoogle Scholar
  4. 4.
    Marshall E. (2002) What to do when clear success comes with an unclear risk? Science 298, 510–511.PubMedCrossRefGoogle Scholar
  5. 5.
    Golzio, M., Teissié, J., and Rols, M. (2001) Control by membrane order of voltage-induced permeabilization, loading and gene transfer in mammalian cells. Bioelectrochemistry 53, 25–34.PubMedCrossRefGoogle Scholar
  6. 6.
    Golzio, M., Teissie, J., and Rols, M. (2002) Direct visualization at the single-cell level of electrically mediated gene delivery. Proc. Natl. Acad. Sci. USA 99, 1292–1297.PubMedCrossRefGoogle Scholar
  7. 7.
    Graham, F. L., and Van der Eb A. J. (1973) A new technique for the assay of infectivity of human adeno-virus 5 DNA. Virology 52, 456467PubMedCrossRefGoogle Scholar
  8. 8.
    Kim, Y., Park, J., Lee, M., Kim, Y., Park, T., and Kim S. (2005) Polyethylenimine with acid-labile linkages as a biodegradable gene carrier. Journal of Controlled Release 103, 209–219.PubMedCrossRefGoogle Scholar
  9. 9.
    Pedraza, C., Bassett, D., McKee, M., Nelea, V., Gbureck, U., and Barralet, J. (2008) The importance of particle size and DNA condensation salt for calcium phosphate nanoparticle transfection. Biomaterials 29, 3384–3392.PubMedCrossRefGoogle Scholar
  10. 10.
    Lasic D. D., Templeton N. S. (1996) Liposomes in gene therapy. Advanced Drug Delivery Reviews, 20, 2–3, 221–266CrossRefGoogle Scholar
  11. 11.
    Lasic, D. (1997) Recent developments in medical applications of liposomes: sterically stabilized liposomes in cancer therapy and gene delivery in vivo. Journal of Controlled Release 48, 203–222.CrossRefGoogle Scholar
  12. 12.
    Dufès, C., Uchegbu, I., and Schätzlein, A. (2005) Dendrimers in gene delivery. Adv. Drug. Deliv. Rev. 57, 2177–2202.PubMedCrossRefGoogle Scholar
  13. 13.
    Shcharbin, D., Klajnert, B., and Bryszewska, M. (2009) Dendrimers in gene transfection. Biochemistry (Mosc) 74, 1070–1079.CrossRefGoogle Scholar
  14. 14.
    Boussif, O., Lezoualc’h, F. Zanta, M., Mergny, M., Scherman, D., Demeneix, B., and Behr, J. (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc. Natl. Acad. Sci. USA 92, 7297–7301.PubMedCrossRefGoogle Scholar
  15. 15.
    Lungwitz, U., Breunig, M., Blunk, T., and Göpferich, A. (2005) Polyethylenimine-based non-viral gene delivery systems. Eur. J. Pharm. Biopharm. 60, 247–266.PubMedCrossRefGoogle Scholar
  16. 16.
    Breunig, M., Lungwitz, U., Liebl, R., Klar, J. Obermayer, B., Blunk, T., and Goepferich, A. (2007) Mechanistic insights into linear polyethylenimine-mediated gene transfer. Biochim. Biophys. Acta 1770, 196–205.PubMedCrossRefGoogle Scholar
  17. 17.
    Kunert, R., Steinfellner, W., Purtscher, M., Assadian A., Katinger, H., (2000) Stable Recombinant Expression of the Anti HIV-1 Monoclonal Antibody 2F5 After IgG3/IgG1 Subclass Switch in CHO Cells. Biotechnology and Bioengineering 67 (1): 97–103PubMedCrossRefGoogle Scholar
  18. 18.
    Gach, JS., Quendler, H., Weik, R., Katinger, H., Kunert, R., (2007) Partial humanisation of an anti-idiotypic antibody against monoclonal antibody 2F5, a potential HIV-1 vaccine? Aids Research and Human Retroviruses; 23 (11), 1405–15PubMedCrossRefGoogle Scholar
  19. 19.
    Alt, F. W., Kellems, R. E., Bertino, J. R., and Schimke, R. T. (1978) Selective multiplication of dihydrofolate reductase genes in methotrexate-resistant variants of cultured murine cells. J. Biol. Chem. 253, 1357–1370.PubMedGoogle Scholar
  20. 20.
    Urlaub, G., and Chasin, L. A. (1980) Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. Proc. Natl. Acad. Sci. USA 77, 4216–4220.PubMedCrossRefGoogle Scholar
  21. 21.
    Zuidam, N., and Barenholz, Y. (1997) Electrostatic parameters of cationic liposomes commonly used for gene delivery as determined by 4-heptadecyl-7-hydroxycoumarin. Biochim. Biophys. Acta 1329, 211–222.PubMedCrossRefGoogle Scholar
  22. 22.
    Simberg, D., Danino, D., Talmon, Y., Minsky, A., Ferrari, M., Wheeler, C., and Barenholz, Y. (2001) Phase behavior, DNA ordering, and size instability of cationic lipoplexes. Relevance to optimal transfection activity. J. Biol. Chem. 276, 47453–47459.Google Scholar
  23. 23.
    D.G. Hunter, D.G. and Frisken, B.J. (1998) Effect of Extrusion Pressure and Lipid Properties on the Size and Polydispersity of Lipid Vesicles. Biophysical Journal, 74, 2996–3002.Google Scholar
  24. 24.
    Reisinger, H., Sevcsik, E., Vorauer-Uhl, K., Lohner, K., Katinger, H., and Kunert, R. (2007) Serum-free transfection of CHO-cells with tailor-made unilamellar vesicles. Cytotechnology 54, 157–168.PubMedCrossRefGoogle Scholar
  25. 25.
    Regelin, A. E., Fankhaenel, S., Gurtesch, L., Prinz, C., von Kiedrowski, G., and Massing, U. (2000) Biophysical and lipofection studies of DOTAP analogs. Biochim. Biophys. Acta 1464, 151–164.PubMedCrossRefGoogle Scholar
  26. 26.
    Reisinger, H., Steinfellner, W., Katinger, H., and Kunert, R. (2009) Serum-free transfection of CHO cells with chemically defined trans­fection systems and investigation of their ­potential for transient and stable transfection. Cytotechnology 60, 115–123.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Department of BiotechnologyUniversity of Natural Resources and Life SciencesViennaAustria

Personalised recommendations