Limiting factors governing protein expression following polyethylenimine-mediated gene transfer in HEK293-EBNA1 cells

Carpentier, Éric Le; Paris, Sébastien; Kamen, Amine; Durocher, Yves

doi:10.1016/j.jbiotec.2006.10.014

Cited by 81 publications

(72 citation statements)

References 47 publications

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“…Interestingly, many large SDC-free polyplexes could be observed in the cytoplasm at 24 hpt in both cases, suggesting that the presence of polyplexes in the cyto- plasm is not sufficient to enable transfection of cells. This is further supported by previous observation showing that plasmid DNA encoding BFP remains in the cytosol for up to 96 hpt in both BFP-expressing and BFP-nonexpressing cells (47). As SDC1 was tagged with GFP at its C-terminal end, we cannot rule out the possibility that polyplexes were still associated with cleaved SDC1 ectodomains at 24 hpt, although the inhibitory effect of SDC2 was not rescued by SDC1 coexpression.…”

Section: Discussionsupporting

confidence: 73%

Opposing Roles of Syndecan-1 and Syndecan-2 in Polyethyleneimine-mediated Gene Delivery

Paris

Burlacu

Durocher

2008

Journal of Biological Chemistry

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Polyethyleneimines (PEIs) are efficient non-viral vectors for gene transfer. Heparan sulfate proteoglycans have been proposed to be the cell-surface receptors for PEI⅐DNA complexes (polyplexes). Here, we investigated if syndecan-1 (SDC1) and syndecan-2 (SDC2) are involved in PEI-mediated transfection. Following addition of polyplexes to HEK293 cells, green fluorescent protein-tagged SDCs rapidly formed clusters with PEI that were dependent of lipid raft integrity. However, although SDC1 overexpression slightly enhanced PEI-mediated gene expression, SDC2 dramatically inhibited it. Confocal microscopy analysis showed that SDC1⅐polyplex endocytosis occurred within minutes after addition of polyplexes, whereas SDC2⅐polyplex endocytosis took hours. Expression of SDC1 cytoplasmic deletion mutants revealed that the SDC1 cytoplasmic tail is required for gene expression, but not for clustering or endocytosis, whereas overexpression of SDC1/SDC2 chimeras showed that the SDC2 ectodomain is responsible for the inhibitory effect on gene transfer. This study provides evidence that SDCs may have opposing effects on PEI-mediated transfection.

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Section: Discussionsupporting

confidence: 73%

Opposing Roles of Syndecan-1 and Syndecan-2 in Polyethyleneimine-mediated Gene Delivery

Paris

Burlacu

Durocher

2008

Journal of Biological Chemistry

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“…As previously observed using the indirect method, LPEI was more efficient than BPEI at transfecting HEK293 cells [7,18] and increasing the concentrations of plasmid and PEI (Fig. 1B) did not increase productivities [31]. A DNA:LPEI ratio of 1:3 with 1 lg/mL of plasmid DNA was thus chosen for the subsequent experiments.…”

Section: Optimization Of Transfection Parameters For the Direct Methodssupporting

confidence: 54%

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

Raymond

Tom

Perret

et al. 2011

Methods

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“…One possible explanation for the lower cell specific productivity could be a difference in transfection efficiency or uptake of the DNA into the nucleus. Carpentier et al (2007) investigated limiting factors governing protein expression following polyethylenimine-mediated gene transfer in HEK293-EBNA1 cells. This study described that transfection efficiencies can vary significantly from one experiment to another even though the same transfection parameters and culture conditions were rigorously applied at all times.…”

Section: Discussionmentioning

confidence: 99%

Development of a generic transient transfection process at 100 L scale

et al. 2008

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We have developed a generic transient transfection process at 100 L scale, using HEK293-EBNA cells and PEI as the transfection reagent for the production of recombinant IgG. The process, including large-scale plasmid preparation, expression at bioreactor scale, capture, purification and, if necessary, endotoxin removal allows reproducible production of more than 0.5 g IgG for in vitro and in vivo studies. We compared the performance of two HEK cell lines, investigated the effect of conditioned medium, optimized the DNA:PEI ratio and implemented a feed strategy to prolong the culture time to increase product yield. The transient transfection protocol developed enables a closed process from seeding culture to protein capture. The challenge of performing a medium exchange before transfection at large scale is solved by applying a continuous centrifugation step between the seeding bioreactor and the production bioreactor. After 7-8 days the harvest and capture is performed in a one-step operation using a Streamline expanded bed chromatography system. Following a polishing step the purified antibody is transferred to the final formulation buffer. The method has shown to be reproducible at 10, 50, and 100 L scale expressing between 5 and 8 mg L -1 IgG.

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Limiting factors governing protein expression following polyethylenimine-mediated gene transfer in HEK293-EBNA1 cells

Cited by 81 publications

References 47 publications

Opposing Roles of Syndecan-1 and Syndecan-2 in Polyethyleneimine-mediated Gene Delivery

Opposing Roles of Syndecan-1 and Syndecan-2 in Polyethyleneimine-mediated Gene Delivery

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

Development of a generic transient transfection process at 100 L scale

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