Study on the method for labeling plasmid DNA with fluorescein / 国际生物医学工程杂志

ABSTRACT

ObjectiveConjugation of fluorescent dye onto plasmid DNA was investigated in order to monitor delivery process of plasmid DNA.MethodsPlasmid was activated with bromine,stored for different timeintervals at 4 ℃ or room temperature,and subsequently coupled with 1,10-diaminodecane to prepare aminemodified plasmid DNA.Amine-modified plasmid was then reacted with isothiocyanate (FITC) for fluorescent labeling,and the labeling ratio was calculated after purification.The effect of storage conditions (time/temperature) of bromine-actived plasmid (BP) on fluorescent labeling efficacy was estimated,and the cell transfection efficiency of fluorescent plasmid-lipofectamine complex was observed.The fluorescent plasmid delivered by lipofectamine 2000 in A10 cells was observed by laser scanning confocal microscope (LSCM) and flow cytometry.ResultsThe experimental data showed that prolonged storage time of bromine-activated DNA had a negative effect on the labeling ratio,and lower storage temperature had a positive effect on the labeling ratio.It also demonstrated that FITC modification had no effect on the transfection efficiency of plasmid-lipofectamine complex as compared with that of unlabeled plasmid-lipofectamine complex,and FITC modified plasmid had enough fluorescent intensity to monitor cell uptake with flow cytometer and sub-cellular distribution with LSCM.ConclusionA facile method for conjugating fluorescent dye onto plasmid was established in the study,and could be utilized to trace the plasmid delivery for investigating the transfection mechanism.