Abstract
The kinetics of polyethylenimine (PEI)-mediated gene transfer at early times after transfection of Chinese hamster ovary (CHO) cell in suspension were investigated using a novel in vitro assay. Addition of an excess of competitor DNA to the culture medium at various times after the initiation of transfection inhibited further cellular uptake of PEI–DNA particles. Using this approach, a constant rate of particle uptake was observed during the first 60 min of transfection at a PEI:DNA ratio of 2:1 (w/w) and a cell density of 2 × 106 cells/ml under serum-free conditions. The uptake rate declined considerably during the next 2 h of transfection. Both the rate and the level of PEI–DNA uptake in serum-free minimal medium were found to be dependent on the PEI–DNA ratio, the cell density at the time of transfection, and the extent of particle aggregation. These studies of the early phase of PEI-mediated transfection are expected to lead to further opportunities for optimization of gene transfer to suspension cultures of mammalian cells for the purpose of large-scale transient recombinant protein production.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Pham, P. L., Kamen, A., & Durocher, Y. (2006). Large-scale transfection of mammalian cells for the fast production of recombinant proteins. Molecular Biotechnology, 34, 225–237. doi:10.1385/MB:34:2:225.
Baldi, L., Hacker, D. L., Adam, M., & Wurm, F. M. (2007). Recombinant protein production by large-scale transient gene expression in mammalian cells: State of the art and future perspectives. Biotechnological Letters, 29, 677–684. doi:10.1007/s10529-006-9297-y.
Boussif, O., Lezoualc’h, F., Zanta, M. A., Mergny, M. D., Scherman, D., Demeneix, B., et al. (1995). A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethylenimine. Proceedings of the National Academy of Sciences of the United States of America, 92, 7297–7301. doi:10.1073/pnas.92.16.7297.
Pollard, H., Remy, J. S., Loussouarn, G., Demolombe, S., Behr, J. P., & Escande, D. (1998). Polyethylenimine but not cationic lipids promotes transgene delivery to the nucleus in mammalian cells. Journal of Biological Chemistry, 273, 7507–7511. doi:10.1074/jbc.273.13.7507.
Godbey, W. T., Wu, K. K., & Mikos, A. G. (1999). Tracking the intracellular path of poly(ethylenimine)/DNA complexes for gene delivery. Proceedings of the National Academy of Sciences of the United States of America, 96, 5177–5181. doi:10.1073/pnas.96.9.5177.
Godbey, W. T., Barry, M. A., Saggau, P., Wu, K. K., & Mikos, A. G. (2000). Poly(ethylenimine)-mediated transfection: a new paradigm for gene delivery. Journal of Biomedical Materials Research, 51, 321–328. doi:10.1002/1097-4636(20000905)51:3<321::AID-JBM5>3.0.CO;2-R.
Choosakoonkriang, S., Lobo, B. A., Koe, G. S., Koe, J. G., & Middaugh, C. R. (2003). Biophysical characterization of PEI/DNA complexes. Journal of Pharmaceutical Sciences, 92, 1710–1722. doi:10.1002/jps.10437.
Demeneix, B., & Behr, J. P. (2005). Polyethylenimine (PEI). Advances in Genetics, 53, 217–230.
Akinc, A., Thomas, M., Klibanov, A., & Langer, R. (2005). Exploring polyethylenime-mediated DNA transfection and the proton sponge hypothesis. Journal of Gene Medicine, 7, 657–663. doi:10.1002/jgm.696.
Bertschinger, M., Schertenleib, A., Backliwal, G., Jordan, M., Hacker, D. L., & Wurm, F. M. (2006). Dissassembly of polyethylenimine-DNA particles in vitro: Implications for polyethylenimine-mediated DNA delivery. Journal of Controlled Release, 116, 96–104. doi:10.1016/j.jconrel.2006.09.006.
Thomas, M., Lu, J. J., Ge, Q., Zhang, C., Chen, J., & Klibanov, A. M. (2005). Full deacylation of polyethylenimine dramatically boosts its gene delivery efficiency and specificity to mouse lung. Proceedings of the National Academy of Sciences of the United States of America, 102, 5679–5684. doi:10.1073/pnas.0502067102.
Wightman, L., Kircheis, R., Rossler, V., Carotta, S., Ruzicka, R., Kursa, M., et al. (2001). Different behavior of branched and linear polyethylenimine for gene delivery in vitro and in vivo. Journal of Gene Medicine, 3, 362–372. doi:10.1002/jgm.187.
Wurm, F., & Bernard, A. (1999). Large-scale transient expression in mammalian cells for recombinant protein production. Current Opinion in Biotechnology, 10, 156–159. doi:10.1016/S0958-1669(99)80027-5.
Bertschinger, M., Burki, C., Backliwal, G., Hacker, D. L., Jordan, M., & Wurm, F. M. (2006). Polyethylenimine-based quality control assay for plasmid DNA. Analytical Biochemistry, 356, 309–311. doi:10.1016/j.ab.2006.05.002.
Muller, N., Girard, P., Hacker, D. L., Jordan, M., & Wurm, F. M. (2005). Orbital shaker technology for the cultivation of mammalian cells in suspension. Biotechnology and Bioengineering, 89, 400–406. doi:10.1002/bit.20358.
Stettler, M., Jaccard, N., Hacker, D., DeJesus, M., Wurm, F. M., & Jordan, M. (2006). New disposable tubes for rapid and precise biomass assessment for suspension cultures of mammalian cells. Biotechnology and Bioengineering, 95, 1228–1233. doi:10.1002/bit.21071.
Derouazi, M., Girard, P., Van Tilborgh, F., Iglesias, K., Muller, N., Bertschinger, M., et al. (2004). Serum-free large-scale transient transfection of CHO cells. Biotechnology and Bioengineering, 87, 537–545. doi:10.1002/bit.20161.
Hunt, L., Jordan, M., DeJesus, M., & Wurm, F. M. (1999). GFP-expressing mammalian cells for fast, sensitive, noninvasive cell growth assessment in a kinetic mode. Biotechnology and Bioengineering, 65, 201–205. doi:10.1002/(SICI)1097-0290(19991020)65:2<201::AID-BIT10>3.0.CO;2-H.
Boeckle, S., von Gersdorff, K., van der Piepen, S., Culmsee, C., Wagner, E., & Ogris, M. (2004). Purification of polyethylenimine polyplexes highlights the role of free polycations in gene transfer. Journal of Gene Medicine, 6, 1102–1111. doi:10.1002/jgm.598.
Kichler, A., Leborgne, C., Coeytaux, E., & Danos, O. (2001). Polyethylenimine-mediated gene delivery: A mechanistic study. Journal of Gene Medicine, 3, 135–144. doi:10.1002/jgm.173.
Fujimoto, L. M., Roth, R., Heuser, J. E., & Schmid, S. L. (2000). Actin assembly plays a variable, but not obligatory role in receptor-mediated endocytosis in mammalian cells. Traffic, 1, 161–171. doi:10.1034/j.1600-0854.2000.010208.x.
Backliwal, G., Hildinger, M., Hasija, V., & Wurm, F. M. (2007). High-density transfection with HEK-293 cells allows doubling of transient titers and removes need for a priori DNA complex formation with PEI. Biotechnology and Bioengineering, 99, 721–727. doi:10.1002/bit.21596.
Bertschinger, M., Chaboche, S., Jordan, M., & Wurm, F. M. (2004). A spectrophotometric assay for the quantification of polyethylenimine in DNA nanoparticles. Analytical Biochemistry, 334, 196–198. doi:10.1016/j.ab.2004.07.020.
Clamme, J. P., Krishnamoorthy, G., & Mely, Y. (2003). Intracellular dynamics of the gene delivery vehicle polyethylenimine during transfection: investigation by two-photon fluorescence correlation spectroscopy. Biochimica et Biophysica Acta, 1617, 52–61. doi:10.1016/j.bbamem.2003.09.002.
Acknowledgments
We thank Dr. Lucia Baldi for critically reading the manuscript. Financial support was provided by the Swiss Innovation Promotion Agency (KTI/CTI).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Bertschinger, M., Schertenleib, A., Cevey, J. et al. The Kinetics of Polyethylenimine-Mediated Transfection in Suspension Cultures of Chinese Hamster Ovary Cells. Mol Biotechnol 40, 136–143 (2008). https://doi.org/10.1007/s12033-008-9069-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12033-008-9069-0