title: Comparative study of polyethylenimines for transient gene expression in mammalian HEK293 and CHO cells.
abstract: Three commercially available linear polyethylenimines (25kDa LPEI, 40kDa PEI"Max" and PEIpro™) were compared regarding their potency to transfect serum-free growing and suspension-adapted HEK293 and CHO cells. We determined the optimal DNA:PEI ratios for maximal expression of the reporter gene SEAP while monitoring cytotoxicity following transfection. PEIs acylation was determined by (1)H NMR and their apparent size and polydispersity assessed by size-exclusion chromatography. The propensity of PEIs to condense plasmid DNA was evaluated by agarose-gel electrophoresis. The zeta potentials and particle sizes at optimal DNA:PEI ratio were analyzed. Polyplex attachment to the cells and internalization kinetics were monitored. The quantity of PEIpro™ needed to efficiently transfect the cells was significantly lower than with LPEI and PEI"Max" and, interestingly, the maximal amount of internalized PEIpro™-based polyplexes was approximately half of that observed with its counterparts. PEIpro™ was the largest and least polydisperse polymer, but also the most cytotoxic. The optimal transfection conditions were subsequently used to express three monoclonal antibodies at larger-scale. The use of the deacylated PEI"Max" and PEIpro™ resulted in a significant increase of recombinant protein expression compared to LPEI. These findings demonstrate the importance of properly choosing the most suitable polymers to obtain optimal recombinant protein transient expression.
authors.affiliation: Département de biochimie et médecine moléculaire, Faculté de Médecine, Université de Montréal, QC, Canada; National Research Council of Canada, Building Montreal-Royalmount, 6100 Royalmount Avenue, Montreal, QC H4P 2R2, Canada.
authors.forename: Laurence
authors.initials: L
authors.lastname: Delafosse
authors.affiliation: National Research Council of Canada, Building Montreal-Royalmount, 6100 Royalmount Avenue, Montreal, QC H4P 2R2, Canada.
authors.forename: Ping
authors.initials: P
authors.lastname: Xu
authors.affiliation: Département de biochimie et médecine moléculaire, Faculté de Médecine, Université de Montréal, QC, Canada; National Research Council of Canada, Building Montreal-Royalmount, 6100 Royalmount Avenue, Montreal, QC H4P 2R2, Canada. Electronic address: yves.durocher@cnrc-nrc.gc.ca.
authors.forename: Yves
authors.initials: Y
authors.lastname: Durocher
mesh_terms: D000215:Acylation; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D016466:CHO Cells; D016923:Cell Death; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000378:metabolism; D004705:Endocytosis; D015870:Gene Expression; D005822:Genetic Vectors / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D008322:Mammals; D011094:Polyethyleneimine / Q000378:metabolism*; D066244:Proton Magnetic Resonance Spectroscopy; D014162:Transfection
chemical_list: D000906:Antibodies; D008164:Luminescent Proteins; C409504:blue fluorescent protein, Aequorea victoria; D011094:Polyethyleneimine; D004247:DNA
keywords: Acylation; Attachment; Internalization; Polyplex; Recombinant protein; Transfection