Biotechnology Letters

, Volume 28, Issue 6, pp 373–382 | Cite as

Generation of Recombinant Chinese Hamster Ovary Cell Lines by Microinjection

  • Madiha Derouazi
  • Rachel Flaction
  • Philippe Girard
  • Maria de Jesus
  • Martin Jordan
  • Florian M. Wurm
Article

Abstract

Microinjection is a gene transfer technique enabling partial control of plasmid delivery into the nucleus or cytoplasm of cultured animal cells. Here this method was used to establish various recombinant mammalian cell lines. The injection volume was estimated by fluorescence quantification of injected fluorescein isothyocynate (FITC)-dextran. The DNA concentration and injection pressure were then optimized for microinjection into the nucleus or cytoplasm using a reporter plasmid encoding the green fluorescent protein (GFP). Nuclear microinjection was more sensitive to changes in these two parameters than was cytoplasmic microinjection. Under optimal conditions, 80–90% of the cells were GFP-positive 1 day after microinjection into the nucleus or the cytoplasm. Recombinant cell lines were recovered following microinjection or calcium phosphate transfection and analyzed for the level and stability of recombinant protein production. In general, the efficiency of recovery of recombinant cell lines and the stability of reporter protein expression over time were higher following microinjection as compared to CaPi transfection. The results demonstrate the feasibility of using microinjection as a method to generate recombinant cell lines.

Keywords

calcium phosphate Chinese hamster ovary cells (CHO) green fluorescent protein (GFP) microinjection recombinant cell lines 

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References

  1. Bode, J, Schlake, T, Iber, M, Schubeler, D, Seibler, J, Snezhkov, E, Nikolaev, L 2000The transgeneticist’s toolbox: novel methods for the targeted modification of eukaryotic genomesBiol. Chem.381801813CrossRefPubMedGoogle Scholar
  2. Brinster, RL, Chen, HY, Trumbauer, ME, Yagle, MK, Palmiter, RD 1985Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggsProc. Natl. Acad. Sci.8244384442PubMedGoogle Scholar
  3. Brunner, S, Sauer, T, Carotta, S, Cotten, M, Saltik, M, Wagner, E 2000Cell cycle dependence of gene transfer by liploplex, polyplex and recombinant adenovirusGene Ther.7401407CrossRefPubMedGoogle Scholar
  4. Capecchi, MR 1980High efficiency transformation by direct microinjection of DNA into cultured mammalian cellsCell22479488CrossRefPubMedGoogle Scholar
  5. Dean, DA 1997Import of plasmid DNA into the nucleus is sequence specificExp. Cell Res.230293302CrossRefPubMedGoogle Scholar
  6. Folger, KR, Wong, EA, Wahl, G, Capecchi, MR 1982Patterns of integration of DNA microinjected into cultured mammalian cells: evidence for homologous recombination between injected plasmid DNA moleculesMol. Cell Biol.213721387PubMedGoogle Scholar
  7. Graham, FL, Eb, AJ 1973A new technique for the essay of the infectivity of human adenovirus 5 DNAVirology52456467CrossRefPubMedGoogle Scholar
  8. Grosjean, F, Batard, P, Jordan, M, Wurm, F 2002S-phase synchronized CHO cells show elevated transfection efficiency and expression using CaPiCytotechnology385762CrossRefGoogle Scholar
  9. Jordan, M, Schallhorn, A, Wurm, FM 1996Transfecting mammalian cells: optimization of critical parameters affecting calcium phosphate precipitate formationNucleic Acids Res.24596601CrossRefPubMedGoogle Scholar
  10. Jordan, M, Wurm, F 2004Transfection of adherent and suspended cells by calcium phosphateMethods33136143CrossRefPubMedGoogle Scholar
  11. Kim, SY, Lee, JH, Shin, HS, Kang, HJ, Kim, YS 2002The human elongation factor 1 alpha (EF-1 alpha) first intron highly enhances expression of foreign genes from the murine cytomegalovirus promoterJ. Biotechnol.93183187CrossRefPubMedGoogle Scholar
  12. Lechardeur, D, Sohn, KJ, Haardt, M, Joshi, PB, Monck, M, Graham, RW, Beatty, B, Squire, J, O’Brodovich, H, Lukacs, GL 1999Metabolic instability of plasmid DNA in the cytosol: a potential barrier to gene transferGene Ther.6482497CrossRefPubMedGoogle Scholar
  13. Manivasakam, P, Aubrecht, J, Sidhom, S, Schiestl, RH 2001Restriction enzymes increase efficiencies of illegitimate DNA integration but decrease homologous integration in mammalian cellsNucleic Acids Res.2948264833CrossRefPubMedGoogle Scholar
  14. Mirzayans, R, Aubin, RA, Paterson, MC 1992Differential expression and stability of foreign genes introduced into human fibroblasts by nuclear versus cytoplasmic microinjectionMutat. Res.281115122CrossRefPubMedGoogle Scholar
  15. Muller, N, Girard, P, Hacker, DL, Jordan, M, Wurm, FM 2005Orbital shaker technology for the cultivation of mammalian cells in suspensionBiotechnol. Bioeng.89400406CrossRefPubMedGoogle Scholar
  16. Nelson, PR, Kent, KC 2002Microinjection of DNA into the nuclei of human vascular smooth muscle cellsJ. Surg. Res.106202208CrossRefPubMedGoogle Scholar
  17. Pepperkok, R, Schneider, C, Philipson, L, Ansorge, W 1988Single cell assay with an automated capillary microinjection systemExp. Cell Res.178369376CrossRefPubMedGoogle Scholar
  18. Pollard, H, Toumaniantz, G, Amos, JL, Avet-Loiseau, H, Guihard, G, Behr, JP, Escande, D 2001Ca2+-sensitive cytosolic nucleases prevent efficient delivery to the nucleus of injected plasmidsJ. Gene Med.3153164CrossRefPubMedGoogle Scholar
  19. Tanaka, AS, Komuro, K 2005Targeted rearrangement of a chromosomal repeat sequence by transfection of a homologous DNA sequence using purified integraseGene Ther.12783794CrossRefPubMedGoogle Scholar
  20. Thomas, M, Klibanov, AM 2003Non-viral gene therapy: polycation-mediated DNA deliveryAppl. Microbiol. Biotechnol.622734CrossRefPubMedGoogle Scholar
  21. Tsulaia, TV, Prokopishyn, NL, Yao, A, Carsrud, ND, Carou, MC, Brown, DB, Davis, BR, Yannariello-Brown, J 2003Glass needle-mediated microinjection of macromolecules and transgenes into primary human mesenchymal stem cellsJ. Biomed. Sci.10328336PubMedGoogle Scholar
  22. Urlaub, G, Kas, E, Carothers, AM, Chasin, LA 1983Deletion of the diploid dihydrofolate reductase locus from cultured mammalian cellsCell33405412CrossRefPubMedGoogle Scholar
  23. Vacik, J, Dean, BS, Zimmer, WE, Dean, DA 1999Cell-specific nuclear import of plasmid DNAGene Ther.610061014CrossRefPubMedGoogle Scholar

Copyright information

© Springer 2006

Authors and Affiliations

  • Madiha Derouazi
    • 1
  • Rachel Flaction
    • 1
  • Philippe Girard
    • 1
  • Maria de Jesus
    • 1
  • Martin Jordan
    • 1
  • Florian M. Wurm
    • 1
  1. 1.Laboratory of Cellular BiotechnologyInstitute of Biological Engineering and BiotechnologyLausanneSwitzerland

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