Studies on the Transfer of DNA into Cells Through Use of Avidin-Polylysine Conjugates Complexed to Biotinylated Transferrin and DNA

A simple procedure has been developed for studying the transfer of DNA into cells using the process of receptor-mediated endocytosis. Poly-L-lysine(460) was covalently attached to the carbohydrate chains of avidin via periodate oxidation and NaBH4 reduction to give avidin-pLys(460). Following purification through Sephacryl WOO, the conjugate was reacted with biotinylated transferrin. The resulting avidin-pLys(460)[bio-transferrin] could be either fractionated by Superose 12 gel chromatography or used directly in experiments with DNA. Determination of the interaction between avidin-pLys(460)[bio-transferrin] and DNA was carried out by nitro cellulose filter binding and agarose gel retardation assays. The conjugate was shown to bind DNA strongly, giving stable complexes soluble in 0.15-0.2 M salt solutions. Gene transfer using avidin-pLys(460)[bio-transferrin] and the luciferase plasmid pRSVL was accomplished with Hela cells, alpha T-3 pituitary cells and a human melanoma cell line used in the present study. Transfection was dependent on bio-transferrin and stimulated by the lysosomotropic agent chloroquine. The results are consistent with a receptor-mediated endocytosis mechanism of DNA delivery for Hela cells and a combination of receptor and adsorptive endocytosis for the alpha T-3 pituitary and melanoma T-5 cell lines.