RESUMEN

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.

Asunto(s)

ADN/química , Colorantes Fluorescentes/química , Naftalenosulfonatos/química , Naftoquinonas/química , Plásmidos/química , Células 3T3 , Animales , Línea Celular , Chlorocebus aethiops , ADN/genética , Colorantes Fluorescentes/síntesis química , Expresión Génica , Colorantes Verde de Lisamina/síntesis química , Colorantes Verde de Lisamina/química , Ratones , Microinyecciones , Naftalenosulfonatos/síntesis química , Naftoquinonas/síntesis química , Fotoquímica , Plásmidos/genética , Plásmidos/metabolismo , Transfección , Transgenes