Transcription of plasmid DNA: influence of plasmid DNA/polyethylenimine complex formation.
Polyethylenimine (PEI) is one of the most potent non-viral vectors. We have developed a lactosylated PEI (Lac-PEI) to enhance cell-specific transfection and have shown that Lac-PEI is more efficient than unsubstituted PEI for gene transfer into immortalized cystic fibrosis airway epithelial SigmaCFTE29o-cells. As both intact PEI/plasmid and Lac-PEI/plasmid complexes are found in the cell nucleus, we have investigated the transcription efficiency of the plasmid complexed with PEI or Lac-PEI, according to the polymer nitrogen/DNA phosphate (N/P) ratio (from 0 to 20). The initiation of transgene transcription was analyzed in an acellular nuclease S1 transcription assay. For both PEI and Lac-PEI complexes, transcription efficiency varied with the N/P ratio of the complexes. Transcription inhibition was observed when plasmid DNA was either loosely (N/P<5) or tightly condensed (N/P>15). For an N/P ratio of 5 and up to 15, transcription of the complexed plasmid was as efficient as that of the free plasmid. Similar results were observed when gene expression was studied after nuclear microinjection of the complexes into SigmaCFTE29o-cells. Our study shows that condensation of DNA influences the accessibility of the plasmid to the transcription machinery. Interestingly, the charge ratios that allow the most efficient transcription are those usually known to be the most efficient for gene transfer in vitro and in vivo.
Honoré
Isabelle
I
Université Paris-Descartes, Faculté de Médecine, AP-HP, Hôpital Cochin, EA 2511, Paris, France.
Grosse
Stéphanie
S
Frison
Natacha
N
Favatier
Florence
F
Monsigny
Michel
M
Fajac
Isabelle
I
D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004247:DNA / Q000235:genetics*; D004563:Electrochemistry; D004587:Electrophoresis, Agar Gel; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007785:Lactose / Q000737:chemistry; D008845:Microinjections; D008854:Microscopy, Electron; D010957:Plasmids / Q000235:genetics*; D011094:Polyethyleneimine / Q000737:chemistry*; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000096:biosynthesis / Q000235:genetics; D014158:Transcription, Genetic
D049452:Green Fluorescent Proteins; D011094:Polyethyleneimine; D004247:DNA; D015719:Single-Strand Specific DNA and RNA Endonucleases; D007785:Lactose