Further studies on targeted DNA transfer to cells using a highly efficient delivery system of biotinylated transferrin and biotinylated polylysine complexed to streptavidin.
Conjugates consisting of biotinylated transferrin and biotinylated poly-L-lysine attached to streptavidin have been prepared and found to transfer luciferase plasmid DNA very efficiently to HeLa cells in the presence of chloroquine. Transfection was dependent on (i) use of biotinylated short chain polylysine containing 70 lysine residues, (ii) biotinylated transferrin containing 1-2 biotin moieties, (iii) reaction of biotinylated transferrin with streptavidin followed by isolation of the resulting conjugate on Sephadex G-200 and (iv) interaction of streptavidin-biotinylated transferrin with biotinylated polylysine giving a complex suitable for DNA transfection. It was found that if the above sequence of steps resulting in the formation of streptavidin-biotinylated transferrin/biotinylated polylysine was followed without isolation of intermediate conjugates by Sephadex G-200 chromatography, pRSVL DNA transfer was still very efficient. Transfer of luciferase DNA by the streptavidin conjugates and subsequent expression of luciferase activity was almost completely inhibited by excess free transferrin, showing that gene transfer was through the transferrin receptor pathway via receptor-mediated endocytosis. The streptavidin (bio2-transferrin) bio10-pLys70 conjugate used in the present experiments was approximately one hundred times more efficient in pRSVL DNA transfection with the HeLa cells than the previously described avidin-pLys460 (bio-transferrin) complex.
Schoeman
R
R
Department of Pharmacology, Faculty of Medicine, University of Stellenbosch, Tygerberg, South Africa.
Joubert
D
D
Ariatti
M
M
Hawtrey
A O
AO
D001426:Bacterial Proteins / Q000378:metabolism*; D001667:Binding, Competitive; D001682:Biological Availability; D001710:Biotin / Q000737:chemistry / Q000378:metabolism*; D005591:Chemical Fractionation; D002738:Chloroquine / Q000494:pharmacology; D002846:Chromatography, Affinity; D004247:DNA / Q000008:administration & dosage* / Q000378:metabolism; D016503:Drug Delivery Systems; D006367:HeLa Cells / Q000187:drug effects; D006801:Humans; D008024:Ligands; D008156:Luciferases / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011107:Polylysine / Q000378:metabolism* / Q000493:pharmacokinetics; D019809:Streptavidin; D014162:Transfection; D014168:Transferrin / Q000378:metabolism* / Q000493:pharmacokinetics
D001426:Bacterial Proteins; D008024:Ligands; D014168:Transferrin; D011107:Polylysine; D001710:Biotin; D002738:Chloroquine; D004247:DNA; D019809:Streptavidin; D008156:Luciferases