Use of the Green Fluorescent Protein as a Quantitative Reporter of Epidermal Growth Factor Receptor-Mediated Gene Delivery

Abstract

We have developed a novel epidermal growth factor (EGF) receptor-mediated gene transfer system to deliver DNA encoding the green fluorescent protein (GFP) to B82 murine fibroblasts transfected with the human EGF receptor gene. Streptavidin was crosslinked to poly-L-lysine, and the product was purified by a combination of affinity and cation exchange chromatography. The crosslinking and purification were confirmed by SDS-PAGE, and the product was shown by agarose gel electrophoresis to bind DNA. EGF was biotinylated, and EGF-biotin, both in the absence and presence of streptavidin-polylysine and DNA, retained the ability to bind to the EGF receptor on B82 cells with high affinity, as demonstrated by radioactive competition binding. EGF linked to polylysine was mixed with a plasmid encoding GFP, and the resulting conjugates were incubated with B82 cells. Quantification by FACS demonstrated EGF receptor-mediated GFP expression efficiency comparable to standard transfection methods. The combination of EGF, a biotin-streptavidin linkage, EGF receptor- transfected cells, and the GFP provides an unusually versatile experimental system for gaining quantitative mechanistic information on molecular conjugate gene delivery from the cell surface to the nucleus.