Apolipoprotein A-I as a Transport Alternative of Green Fluorescent Protein GFP Gene in Rat Hepatocytes

Abstract

The purpose of this work was to study the possibility of using apolipoprotein A-I (apo A-I) as a transport variant form of green fluorescent protein (GFP) gene in rat hepatocytes. Cultured rat hepatocytes were used as a model. The conjugate of apo A-I with fluorescein isothiocyanate (FITC) was prepared by incubation of the apo A-I protein with FITC in carbonate buffer, pH 9.5, at a ratio of 1.5 μg of FITC per 1 mg of the protein. Plasmids for pE-GAG transfection with an inserted GFP gene were enriched in the promoter region with cis-elements of CC(GCC)3-5 type to enhance complex formation with apo A-I. An inverted fluorescence microscope was used to visually analyze cell fluorescence. The work presents evidence of penetration of FITC-labeled apo A-I into the cytoplasm and nuclei of rat hepatocytes through receptor-mediated endocytosis. Based on this evidence, it was attempted to use apo A-I as a an agent of targeted delivery of plasmid DNA with an inserted GFP gene into a cell. According to the results of fluorescence microscopy, using apo A-I as a transfection agent for plasmid DNA resulted in accumulation of GFP protein in the cytoplasm of hepatocytes. Expression of the GFP gene and accumulation of fluorescent protein were not observed in the absence of apo A-I. The results obtained may indicate the delivery of GFP gene to the nuclear apparatus of the cell, its expression and synthesis of GFP protein.