title: Improved non-viral transfection of glial and adult neural stem cell lines and of primary astrocytes by combining agents with complementary modes of action.
abstract: BACKGROUND
  Rational design of gene vectors for therapeutic applications requires understanding of transfection mechanisms. In this study, multiple transfection assays revealed complementary mechanisms between two commonly used transfection agents. This finding was then exploited to produce improved transfection outcomes.
  
  
  RESULTS
  Rat C6 glial cells, adult rat hippocampal progenitor cells and primary astrocytes were transfected using Lipofectamine (LA) or polyethylenimine (PEI), in vitro. Although LA- and PEI-transfected populations expressed the same total level of transgene product, LA transfected considerably more cells than PEI (approximately 20 vs. 14%). A fluorescently labelled plasmid and time-course analysis, involving both flow cytometry and confocal microscopy, were used to explain this apparent discrepancy. Results showed that LA delivered more plasmid DNA to the cytoplasm and achieved transgene expression in more cells than PEI. In contrast, PEI transfected fewer cells but, on average, produced more transgene product per transfected cell.
  
  
  CONCLUSIONS
  A comparative transfection model was developed to explain these different characteristics. According to this model, transfection is a multistage process with different transfection agents exerting their primary effect at different stages in this process. This model forecast that it should be possible to prepare a chimeric complex with a transfection efficiency that exceeded that achievable with Lipofectamine or polyethylenimine alone. This prediction was tested and shown to hold for glioma cells, primary astrocytes, and adult neural stems cells.
authors.affiliation: Centre for Neuroscience, Flinders Medical Research Institute, Flinders University of South Australia, Adelaide, Australia.
authors.forename: Rogan B
authors.initials: RB
authors.lastname: Tinsley
authors.forename: Melanie J
authors.initials: MJ
authors.lastname: Vesey
authors.forename: Shahram
authors.initials: S
authors.lastname: Barati
authors.forename: Robert A
authors.initials: RA
authors.lastname: Rush
authors.forename: Ian A
authors.initials: IA
authors.lastname: Ferguson
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000187:drug effects*; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D004347:Drug Interactions; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D006624:Hippocampus / Q000166:cytology; D008055:Lipids / Q000494:pharmacology*; D009457:Neuroglia / Q000187:drug effects*; D009474:Neurons / Q000187:drug effects*; D010957:Plasmids / Q000235:genetics; D011094:Polyethyleneimine / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D051381:Rats; D013234:Stem Cells / Q000187:drug effects*; D014162:Transfection / Q000379:methods*; D019076:Transgenes; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D008055:Lipids; C086724:Lipofectamine; D049452:Green Fluorescent Proteins; D011094:Polyethyleneimine; D001616:beta-Galactosidase