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-pLys460. Following purification through Sephacryl S-300, the conjugate was reacted with biotinylated transferrin. The resulting avidin-pLys460-[bio-transferrin] could be either fractionated by Superose 12 gel chromatography or used directly in experiments with DNA. Determination of the interaction between avidin-pLys460-]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-pLys460-[bio-transferrin] and the luciferase plasmid pRSVL was accomplished with Hela cells, alpha T3 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 T3 pituitary and melanoma T-5 cell lines.
Strydom
S
S
Department of Pharmacology, Faculty of Medicine, University of Stellenbosch, Tygerberg, South Africa.
Van Jaarsveld
P
P
Van Helden
E
E
Ariatti
M
M
Hawtrey
A
A
D001360:Avidin / Q000737:chemistry / Q000378:metabolism*; D001710:Biotin / Q000737:chemistry / Q000378:metabolism*; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D004705:Endocytosis / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008546:Melanoma, Experimental / Q000378:metabolism; D011107:Polylysine / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection; D014168:Transferrin / Q000737:chemistry / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D014168:Transferrin; D001360:Avidin; D011107:Polylysine; D001710:Biotin; D004247:DNA; D008156:Luciferases