Microinjection pp 99-112

Part of the Methods in Molecular Biology book series (MIMB, volume 518) | Cite as

DNA Delivery by Microinjection for the Generation of Recombinant Mammalian Cell Lines

  • Sebastien Chenuet
  • Madiha Derouazi
  • David Hacker
  • Florian Wurm
Protocol

Abstract

Gene transfer methods for producing recombinant cell lines are often not very efficient. One reason is that the recombinant DNA is delivered into the cell cytoplasm and only a small fraction reaches the nucleus. This chapter describes a method for microinjecting DNA directly into the nucleus. Direct injection has several advantages including the ability to deliver a defined copy number into the nucleus, the avoidance of DNAses that are present in the cell cytoplasm, and the lack of a need for extensive subcloning to find the recombinant cells. The procedure is described for two cell lines, CHO DG44 and BHK-21, using green fluorescent protein as a reporter gene. However, this method could easily be adapted to other cells lines and using other recombinant genes.

Key words

Recombinant cell line microinjection green fluorescent protein transfection gene expression 

References

  1. 1.
    Coonrod A., Li F.Q., Horwitz M. (1997) On the mechanism of DNA transfection: efficient gene transfer without viruses. Gene Ther. 4, 1313–1321.CrossRefGoogle Scholar
  2. 2.
    James M.B., Giorgio T.D. (2000) Nuclear-associated plasmid, but not cell-associated plasmid, is correlated with transgene expression in cultured mammalian cells. Mol. Ther. 1, 339–346.CrossRefGoogle Scholar
  3. 3.
    Lechardeur D., Sohn K.J., Haardt M., Joshi P.B., Monck M., Graham R.W., Beatty B., Squire J., O'Brodovich H., Lukacs G.L. (1999) Metabolic instability of plasmid DNA in the cytosol: a potential barrier to gene transfer. Gene Ther. 6, 482–497.CrossRefGoogle Scholar
  4. 4.
    Lewis P.F., Emerman M. (1994) Passage through mitosis is required for oncoretroviruses but not for the human immunodeficiency virus. J. Virol. 68, 510–516.Google Scholar
  5. 5.
    Miller D.G., Adam M.A., Miller A.D. (1990) Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. Mol. Cell. Biol. 10, 4239–4242.Google Scholar
  6. 6.
    Mortimer I., Tam P., MacLachlan I., Graham R.W., Saravolac E.G., Joshi P.B. (1999) Cationic lipid-mediated transfection of cells in culture requires mitotic activity. Gene Ther. 6, 403–411.CrossRefGoogle Scholar
  7. 7.
    Wilke M., Fortunati E., van den Broek M., Hoogeveen A.T., Scholte B.J. (1996) Efficacy of a peptide-based gene delivery system depends on mitotic activity. Gene Ther. 3, 1133–1142.Google Scholar
  8. 8.
    Ludtke J.J., Sebestyen M.G., Wolff J.A. (2002) The effect of cell division on the cellular dynamics of microinjected DNA and dextran. Mol. Ther. 5, 579–588.CrossRefGoogle Scholar
  9. 9.
    Ludtke J.J., Zhang G., Sebestyen M.G., Wolff J.A. (1999) A nuclear localization signal can enhance both the nuclear transport and expression of 1 kb DNA. J Cell Sci. 112, 2033–2041.Google Scholar
  10. 10.
    Derouazi M., Flaction R., Girard P., de Jesus M., Jordan M., Wurm F.M. (2006) Generation of recombinant Chinese hamster ovary cell lines by microinjection. Biotechnol. Lett. 28, 373–382.CrossRefGoogle Scholar
  11. 11.
    Brunner S., Sauer T. Carotta S., Cotten M., Saltik M., Wagner E. (2000) Cell cycle dependence of gene transfer by lipoplex, polyplex and recombinant adenovirus. Gene Ther. 7, 401–407.CrossRefGoogle Scholar
  12. 12.
    Escriou V., Ciolina C., Lacroix F., Byk G., Scherman D., Wils P. (1998) Cationic lipid-mediated gene transfer: effect of serum on cellular uptake and intracellular fate of lipopolyamine/DNA complexes. Biochim. Biophys. Acta 1368, 276–288.CrossRefGoogle Scholar
  13. 13.
    Grosjean F., Batard P., Jordan M., Wurm F.M. (2002) S-phase synchronized CHO cells show elevated transfection efficiency and expression using CaPi. Cytotechnology 38, 57–62.CrossRefGoogle Scholar
  14. 14.
    Pollard H., Toumaniantz G., Amos J.L., Avet-Loiseau H., Guihard G., Behr J.P., Escande D. (2001) Ca2+-sensitive cytosolic nucleases prevent efficient delivery to the nucleus of injected plasmids. J. Gene Med. 3, 153–164.CrossRefGoogle Scholar
  15. 15.
    Wolff J.A., Budker V. (2005) The mechanism of naked DNA uptake and expression. Adv. Genet. 54, 3–20.Google Scholar
  16. 16.
    Capecchi M.R. (1981) High efficiency transformation by direct microinjection of DNA into cultured mammalian cells. Cell. 22, 479–488.CrossRefGoogle Scholar
  17. 17.
    Folger K.R., Wong E.A., Wahl G., Capecchi M.R. (1982) Patterns of integration of DNA microinjected into cultured mammalian cells: evidence for homologous recombination between injected plasmid DNA molecules. Mol. Cell. Biol. 2, 1372–1387.Google Scholar
  18. 18.
    Dean D.A., Dean B.S., Muller S., Smith L.C. (1999) Sequence requirements for plasmid nuclear import. Exp. Cell. Res. 253, 713–722.CrossRefGoogle Scholar
  19. 19.
    Muller N., Girard P., Hacker D.L., Jordan M., Wurm F.M. (2005) Orbital shaker technology for the cultivation of mammalian cells in suspension. Biotechnol. Bioeng. 89, 400–406.CrossRefGoogle Scholar
  20. 20.
    Hacker D.L., Derow E., Wurm F.M. (2005) The CELO adenovirus Gam1 protein enhances transient and stable recombinant protein expression in Chinese hamster ovary cells. J Biotechnol. 117, 21–29.CrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Sebastien Chenuet
    • 1
  • Madiha Derouazi
    • 1
  • David Hacker
    • 1
  • Florian Wurm
    • 1
  1. 1.École Polytechnique Féderale de LausanneEPFL-SV-IBI-LBTCLausanneSwitzerland

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