RESUMEN
BACKGROUND: Cationic lipid-mediated gene transfer is a promising approach for gene therapy. However, despite the significant amount of lipoplexes internalized by target cells, transgene expression remains too low. Obstacles to nuclear accumulation of plasmid DNA include: the passage of DNA across the cellular membrane, the dismantling of nucleolipidic particles in the cytoplasm and the nuclear import of plasmid DNA. The purpose of the present study was to evaluate the impact of cell status on cationic lipid-mediated transfer. METHODS: Cells were either growth-arrested (by aphidicolin) or synchronized (by a classical double-thymidine block protocol) and cationic lipid-mediated transfection of these cells was evaluated. For the study of the nuclear import of plasmid DNA, two techniques were developed: microinjection of plasmid DNA into intact cells, and the use of cells permeabilized with digitonin. RESULTS: When CV-1 cells were growth-arrested by aphidicolin, cationic lipid-mediated gene transfer was inhibited. Hela cells were synchronized and incubated with lipoplexes at different times after release of the block. Gene expression was greatly enhanced when cells underwent mitosis. When transfection was performed during the early period after block release, when fewer than 5% of the cells had divided, gene expression was carefully quantified and could be attributed to cells that escaped cell cycle block. However, by direct analysis of nuclear import of GFP-coding plasmid using cytoplasmic microinjection, GFP expression could be detected in a few cells that had not divided. CONCLUSIONS: Cationic lipid-mediated gene transfer efficiency increased when cells underwent mitosis. However, when cells did not divide, gene transfer was not completely abolished. Nuclear import of plasmid was greatly facilitated by a mitotic event. In non-mitotic cells, nuclear envelope crossing by plasmid DNA could be detected but was a very rare event.
Asunto(s)
Núcleo Celular/metabolismo , Lípidos/farmacología , Plásmidos/metabolismo , Transfección , Animales , Afidicolina/farmacología , División Celular/efectos de los fármacos , Línea Celular , Chlorocebus aethiops , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Proteínas Luminiscentes/genética , MicroinyeccionesRESUMEN
We have developed a chemical strategy for covalent coupling of fluorophores to plasmid DNA. A p-azido-tetrafluoro-benzyl-lissamine conjugate was synthesized and purified. This conjugate was used to covalently associate fluorescent molecules to plasmid DNA by photoactivation. In contrast to nick-translated plasmid DNA, plasmid-lissamine conjugates appeared on gel as supercoiled DNA. Reporter gene was expressed after transfection of the plasmid-lissamine conjugates in NIH 3T3 cells, although gene transfer efficiency was decreased by 60% as compared with unlabeled DNA. Intracellular traffic of plasmid-lissamine conjugates was studied in transfected cells. After cytoplasmic microinjection, fluorescent plasmid did not diffuse from the site of injection and appeared to be progressively degraded in the cytoplasm.