Role of intracellular cationic liposome-DNA complex dissociation in transfection mediated by cationic lipids.
The cationic lipid-mediated gene transfer process involves sequential steps: internalization of the cationic lipid-DNA complexes inside the cells via an endocytosis-like mechanism, escape from endosomes, dissociation of the complex, and finally entry of free DNA into the nucleus. However, cationic lipid-DNA complex dissociation in the cytoplasm and the ability of the subsequently released DNA to enter the nucleus have not yet been demonstrated. In this report we showed, using confocal laser scanning analysis, that microinjection of a double fluorescent-labeled cationic lipid-pCMV-LacZ plasmid complex into the cytoplasm of HeLa cells results in efficient complex dissociation. However, the released DNA did not enter the nucleus, and no significant transfection could be detected. In contrast, nuclear microinjection of the cationic lipid-pCMV-LacZ plasmid complex resulted in efficient complex dissociation and transfection of all the cells. Taken together, the data suggest that intracellular dissociation of the cationic lipid-DNA complex is not a limiting step for transfection as previously thought.
Cornelis
Sabine
S
Laboratoire de Structure et Fonction des Membranes Biologiques, Université Libre de Bruxelles, Brussels, Belgium.
Vandenbranden
Michel
M
Ruysschaert
Jean-Marie
JM
Elouahabi
Abdelatif
A
D000578:Amidines / Q000008:administration & dosage / Q000378:metabolism; D001692:Biological Transport; D002412:Cations / Q000008:administration & dosage / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004274:DNA, Recombinant / Q000008:administration & dosage* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000032:analysis; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D007763:Lac Operon; D008081:Liposomes / Q000008:administration & dosage / Q000378:metabolism; D046911:Macromolecular Substances; D008845:Microinjections; D018613:Microscopy, Confocal; D010714:Phosphatidylethanolamines / Q000008:administration & dosage / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014162:Transfection / Q000379:methods*; D001616:beta-Galactosidase / Q000096:biosynthesis
D000578:Amidines; D002412:Cations; D004274:DNA, Recombinant; D005456:Fluorescent Dyes; D008081:Liposomes; D046911:Macromolecular Substances; C089665:N-t-butyl-n'-tetradecyl-3-tetradecylaminopropionamidine; D010714:Phosphatidylethanolamines; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase