| Téléchargement | - Voir le manuscrit accepté : A simplified polyethylenimine-mediated transfection process for large-scale and high-throughput applications (PDF, 628 Kio)
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| DOI | Trouver le DOI : https://doi.org/10.1016/j.ymeth.2011.04.002 |
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| Auteur | Rechercher : Raymond, Celine; Rechercher : Tom, Roseanne1; Rechercher : Perret, Sylvie1; Rechercher : Moussouami, Pascal; Rechercher : L'Abbe, Denis1; Rechercher : St-Laurent, Gilles1; Rechercher : Durocher, Yves1 |
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| Affiliation | - Conseil national de recherches du Canada. Institut de recherche en biotechnologie du CNRC
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| Format | Texte, Article |
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| Sujet | Antibodies; biotechnology; Bioreactor; Bioreactors; Biotechnology; Canada; Cell Line; Cells; Dna; EBNA1; Gene Expression; HEK293; Large-scale production; Nanoparticles; Polyethylenimine; Protein; Proteins; Recombinant protein; Recombinant Proteins; Scopus; Serum-free; Suspension culture; Transfection |
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| Résumé | Transient gene expression in mammalian cells is a valuable alternative to stable cell lines for the rapid production of large amounts of recombinant proteins. While the establishment of stable cell lines takes 2–6 months, milligram amounts of protein can be obtained within a week following transfection. The polycation polyethylenimine (PEI) is one of the most utilized reagents for small- to large-scale transfections as it is simple to use and, when combined with optimized expression vectors and cell lines, provides high transfection efficiency and titers. As with most transfection reagents, PEI-mediated transfection involves the formation of nanoparticles (polyplexes) which are obtained by its mixing with plasmid DNA. A short incubation period that allows polyplexes to reach their optimal size is performed prior to their addition to the culture. As the quality of polyplexes directly impacts transfection efficiency and productivity, their formation complicates scalability and automation of the process, especially when performed in large-scale bioreactors or small-scale high-throughput formats. To avoid variations in transfection efficiency and productivity that arise from polyplexes formation step, we have optimized the conditions for their creation directly in the culture by the consecutive addition of DNA and PEI. This simplified approach is directly transferable from suspension cultures grown in 6-well plates to shaker flasks and 5-L WAVE bioreactors. As it minimizes the number of steps and does not require an incubation period for polyplex formation, it is also suitable for automation using static cultures in 96-well plates. This “direct” transfection method thus provides a robust platform for both high-throughput expression and large-scale production of recombinant proteins. |
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| Date de publication | 2011-12-30 |
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| Dans | |
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| Langue | anglais |
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| Publications évaluées par des pairs | Oui |
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| Numéro du CNRC | NRCC 52782 |
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| Numéro NPARC | 18335886 |
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| Exporter la notice | Exporter en format RIS |
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| Signaler une correction | Signaler une correction (s'ouvre dans un nouvel onglet) |
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| Identificateur de l’enregistrement | 403d9b7f-dde2-4df0-9e53-bf9f1c156f73 |
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| Enregistrement créé | 2011-07-29 |
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| Enregistrement modifié | 2020-04-21 |
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