Limiting factors governing protein expression following polyethylenimine-mediated gene transfer in HEK293-EBNA1 cells.
Transient gene expression in mammalian cells is intensively used for the rapid generation of recombinant proteins for biochemical, biophysical and pre-clinical studies. Still, the principles behind DNA transfer to the cells and the cellular cascade of events that ultimately dictate protein expression levels are not fully understood. Using polyethylenimine (PEI) mediated transfection of HEK293-EBNA1 cells, we sought to determine the most critical parameters that drive and limit recombinant protein production. Our results showed that a maximum of 65,000 plasmid copies/cell can be recovered in total extracts at 1 day post-transfection. Analyses performed after cell sorting revealed equal amounts of plasmid DNA in GFP-positive and -negative populations. However, nuclear plasmid content was three-fold higher in GFP-positive cells (1850 copies) than in GFP-negative cells (550 copies). The fact that significant amounts of plasmid DNA are found in the nucleus of GFP-negative cells suggests that its transcriptional competency is impaired. Interestingly, transfecting cells using a wide range of plasmid quantities at the optimal DNA:PEI ratio did not significantly affect the number of expressing cells. Thus, it appears that successful transgene expression is more likely to depend on a cellular "competent" state than to the quantity of plasmid DNA delivered per cell. Moreover, Northern blot analysis and SEAP/GFP measurement following plasmid titration experiments showed that transcriptional and translational processes are operating near to saturation under optimal transfection conditions. Overall, our results suggest that events that regulate nuclear translocation of plasmid DNA and its transcriptional competency as well as translational/post-translational limitations represent major bottlenecks in the success of a PEI-mediated protein production.
Carpentier
Eric
E
Animal Cell Technology Group, Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Avenue, Montreal, Quebec, Canada H4P 2R2.
Paris
Sébastien
S
Kamen
Amine A
AA
Durocher
Yves
Y
D015152:Blotting, Northern; D002460:Cell Line; D019309:Epstein-Barr Virus Nuclear Antigens / Q000378:metabolism; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D017930:Genes, Reporter / Q000502:physiology*; D005822:Genetic Vectors / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000196:embryology; D010957:Plasmids / Q000235:genetics*; D011094:Polyethyleneimine / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D014162:Transfection / Q000379:methods*; D019076:Transgenes
D019309:Epstein-Barr Virus Nuclear Antigens; D012333:RNA, Messenger; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D011094:Polyethyleneimine; C417029:EBV-encoded nuclear antigen 1