Transient transfection of CHO cells using linear polyethylenimine is a simple and effective means of producing rainbow trout recombinant IFN-γ protein

Cytotechnology - Tập 67 - Trang 987-993 - 2014
Ronggai Li1
1Scottish Fish Immunology Research Centre, University of Aberdeen, Aberdeen, UK

Tóm tắt

A practical method was developed for the transient transfection of Chinese hamster ovary (CHO) cells with 25 kDa linear polyethylenimine (PEI) then optimal culture conditions determined for the production of rainbow trout (Oncorhynchus mykiss) IFN-γ recombinant protein. We found that culture temperature had a significant impact upon recombinant protein yield, with best results being obtained at 32 °C. However the amount of serum added to the culture medium had no effect upon recombinant IFN-γ (rIFN-γ) production. In this study maximal rIFN-γ yields and minimal PEI toxicity were achieved using a DNA/PEI ratio of 1:8, where the amount of PEI did not exceed 10 µg per 5 ml of RPMI1640 culture medium, with cells subsequently cultured at 32 °C for 7 days. Thus, linear PEI is a technically simple and cost-efficient method for the transient transfection of CHO cells and is compatible with serum-free operations.

Tài liệu tham khảo

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. J Gene Med 6:1102–1111 Boussif O, Lezoualc’h F, Zanta MA, Mergny MD, Scherman D, Demeneix B, Behr JP (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 Castro R, Martin SA, Zou J, Secombes CJ (2010) Establishment of an IFN-γ specific reporter cell line in fish. Fish Shellfish Immunol 28:312–319 Demain AL, Vaishnav P (2009) Production of recombinant proteins by microbes and higher organisms. Biotechnol Adv 27:297–306 Derouazi M, Girard P, Van Tilborgh F, Iglesias K, Muller N, Bertschinger M, Wurm FM (2004) Serum-free large-scale transient transfection of CHO cells. Biotechnol Bioeng 87:537–545 Fox SR, Tan HK, Tan MC, Wong SC, Yap MG, Wang DI (2005a) A detailed understanding of the enhanced hypothermic productivity of interferon-gamma by Chinese-hamster ovary cells. Biotechnol Appl Biochem 41:255–264 Fox SR, Yap MX, Yap MG, Wang DI (2005b) Active hypothermic growth: a novel means for increasing total interferon-gamma production by Chinese-hamster ovary cells. Biotechnol Appl Biochem 41:265–272 Galbraith DJ, Tait AS, Racher AJ, Birch JR, James DC (2006) Control of culture environment for improved polyethylenimine-mediated transient production of recombinant monoclonal antibodies by CHO cells. Biotechnol Prog 22:753–762 Hwang SJ, Yoon SK, Koh GY, Lee GM (2011) Effects of culture temperature and pH on flag-tagged COMP angiopoietin-1 (FCA1) production from recombinant CHO cells: FCA1 aggregation. App Microbiol Biotechnol 91:305–315 Kircheis R, Wightman L, Wagner E (2001) Design and gene delivery activity of modified polyethylenimines. Adv Drug Deliv Rev 53:341–358 Klinman DM, McKearn TJ (1981) Dialyzable serum components can support the growth of hybridoma cell lines in vitro. J Immunol Methods 42:1–9 Matasci M, Hacker DL, Baldi L, Wurm FM (2008) Recombinant therapeutic protein production in cultivated mammalian cells: current status and future prospects. Drug Discov Today Technol 5:e37–e42 Miller CH, Maher SG, Young HA (2009) Clinical use of interferon-gamma. Ann N Y Acad Sci 1182:69–79 Muir A, Sylvestre P, Rockey D (2006) Interferon gamma-1b for the treatment of fibrosis in chronic hepatitis C infection. J Viral Hepat 13:322–328 O’Callaghan PM, James DC (2008) Systems biotechnology of mammalian cell factories. Brief Funct Genomic Proteomic 7:95–110 Reed SE, Staley EM, Mayginnes JP, Pintel DJ, Tullis GE (2006) Transfection of mammalian cells using linear polyethylenimine is a simple and effective means of producing recombinant adeno-associated virus vectors. J Virol Methods 138:85–98 Sareneva T, Pirhonen J, Cantell K, Julkunen I (1995) N-glycosylation of human interferon-gamma: glycans at Asn-25 are critical for protease resistance. Biochem J 308:9–14 Schatz SM, Kerschbaumer RJ, Gerstenbauer G, Kral M, Dorner F, Scheiflinger F (2003) Higher expression of Fab antibody fragments in a CHO cell line at reduced temperature. Biotechnol Bioeng 84:433–438 Wightman L, Kircheis R, Rössler V, Carotta S, Ruzicka R, Kursa M, Wagner E (2001) Different behavior of branched and linear polyethylenimine for gene delivery in vitro and in vivo. J Gene Med 3:362–372 Wurm F, Bernard A (1999) Large-scale transient expression in mammalian cells for recombinant protein production. Curr Opin Biotechnol 10:156–159 Zhang Y (2009) Approaches to optimizing animal cell culture process: substrate metabolism regulation and protein expression improvement. In Biotechnology in China I, Springer, Berlin, pp 177–215 Zou J, Carrington A, Collet B, Dijkstra JM, Yoshiura Y, Bols N, Secombes C (2005) Identification and bioactivities of IFN-γ in rainbow trout oncorhynchus mykiss: the first Th1-type cytokine characterized functionally in fish. J Immunol 175:2484–2494