A simplified polyethylenimine-mediated transfection process for large-scale and high-throughput applications

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DOIResolve DOI: http://doi.org/10.1016/j.ymeth.2011.04.002
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TypeArticle
Journal titleMethods
Volumex
Issuex
Pages18; # of pages: 8
SubjectAntibodies; article; bio; 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
AbstractTransient 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.
Publication date
LanguageEnglish
AffiliationNRC Biotechnology Research Institute; National Research Council Canada
Peer reviewedYes
NRC number52782
NPARC number18335886
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Record identifier403d9b7f-dde2-4df0-9e53-bf9f1c156f73
Record created2011-07-29
Record modified2017-03-23
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