Abstract
We describe a method for polyethyleneimine (PEI) mediated transient transfection of suspension-adapted Chinese hamster ovary (CHO-DG44) cells for protein expression applicable at scales from 2 mL to 2 L. The method involves transfection at a high cell density (5 × 106 cells/mL) by direct addition of plasmid DNA (pDNA) and PEI to the culture and subsequent incubation at 31 °C with agitation by orbital shaking. This method requires 0.3 mg/L of coding pDNA, 2.7 mg/L of nonspecific (filler) DNA and 15 mg/L of PEI. The production phase is performed at 31 °C in the presence of 0.25% N,N-dimethylacetamide (DMA). We also provide information on culture vessel options, recommended working volumes, and recommended shaking speeds for transfections at scales from 2 mL to 2 L.
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References
Daramola O, Stevenson J, Dean G, Hatton D, Pettman G, Holmes W, Field R (2014) A high-yielding CHO transient system: coexpression of genes encoding EBNA-1 and GS enhances transient protein expression. Biotechnol Prog 30:132–141
Rajendra Y, Hougland MD, Alam R, Morehead TA, Barnard GC (2015) A high cell density transient transfection system for therapeutic protein expression based on a CHO GS-knockout cell line: process development and product quality assessment. Biotechnol Bioeng 112:977–986
Jain NK, Barkowski-Clark S, Altman R, Johnson K, Sun F, Zmuda J, Liu CY, Kita A, Schulz R, Neill A, Ballinger R (2017) A high density CHO-S transient transfection system: comparison of ExpiCHO and Expi293. Protein Expr Purif 134:38–46
Rajendra Y, Kiseljak D, Baldi L, Hacker DL, Wurm FM (2011) A simple high-yielding process for transient gene expression in CHO cells. J Biotechnol 153:22–26
Rajendra Y, Kiseljak D, Manoli S, Baldi L, Hacker DL, Wurm FM (2012) Role of non-specific DNA in reducing coding DNA requirement for transient gene expression with CHO and HEK-293E cells. Biotechnol Bioeng 109:2271–2278
Kichler A, Leborgne C, Danos O (2005) Dilution of reporter gene with stuffer DNA does not alter the transfection efficiency of polyethylenimines. J Gene Med 7:1459–1467
Rajendra Y, Balasubramanian S, Kiseljak D, Baldi L, Wurm FM, Hacker DL (2015) Enhanced plasmid DNA utilization in transiently transfected CHO-DG44 cells in the presence of polar solvents. Biotechnol Prog 31:1571–1578
Rajendra Y, Hougland MD, Schmitt MG, Barnard GC (2015) Transcriptional and post-transcriptional targeting for enhanced transient gene expression in CHO cells. Biotechnol Lett 37:2379–2386
Muller N, Girard P, Hacker DL, Jordan M, Wurm FM (2005) Orbital shaker technology for the cultivation of mammalian cells in suspension. Biotechnol Bioeng 89:400–406
Klockner W, Buchs J (2012) Advances in shaking technologies. Trends Biotechnol 30:307–314
Duetz WA (2007) Microtiter plates as mini-bioreactors: miniaturization of fermentation methods. Trends Microbiol 15:469–475
Rajendra Y, Balasubramanian S, Hacker DL (2017) Large-scale transient transfection of Chinese hamster ovary cells in suspension. Methods Mol Biol 1603:45–55
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Rajendra, Y. (2018). PEI-Mediated Transient Gene Expression in CHO Cells. In: Hacker, D. (eds) Recombinant Protein Expression in Mammalian Cells. Methods in Molecular Biology, vol 1850. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8730-6_3
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DOI: https://doi.org/10.1007/978-1-4939-8730-6_3
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