Targeted transfer of polyethylenimine-avidin-DNA bioconjugates to hematopoietic cells using biotinylated monoclonal antibodies.
Here we examine whether attachment of biotinylated antibodies to proteins on the cell surface increases the transfection efficiency of polyethylenimine-avidin-DNA bioconjugate gene transfer. Preliminary experiments were performed to compare avidin endocytosis into cells incubated with biotinylated antibodies. Antibody biotinylation resulted in the endocytosis of avidin-FITC into nearly 100% of cells compared with no detectable binding or entry into unbiotinylated cells. Gene transfer was accomplished with avidin conjugated to polyethylenimine (PEI) at a molar ratio of 4:1 (PA4). Plasmid DNA encoding the green fluorescent protein (GFP) gene was condensed on the PA4, and transfection efficiencies were measured by flow cytometry as the percentage of cells that fluoresced at levels greater than two standard deviations above the negative control. Gene transfer efficiencies were compared among K562, HEL, and Jurkat leukemia cell lines. Control transfections with DNA alone or untargeted PEI-DNA resulted in </=2% GFP positive cells. Targeting PEI-avidin-DNA to antibody biotinylated cells increased transfection efficiency several fold over untargeted PEI. For each cell type, the increase in transfection efficiency was not significantly different among four biotinylated antibodies tested (antiCD55, antiCD59, antiCD71, and antiCD98). These data suggest biotinylated antibodies may be useful for targeting polyethylenimine-avidin mediated gene transfer.
Wojda
U
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Laboratory of Chemical Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 9000 Rockville Pike, Bethesda, Maryland 20892, USA.
Miller
J L
JL
D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000954:Antigens, Surface; D001360:Avidin / Q000378:metabolism*; D019921:Biotinylation; D018957:CD59 Antigens / Q000276:immunology; D004247:DNA / Q000378:metabolism*; D004705:Endocytosis; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D020014:K562 Cells; D011094:Polyethyleneimine / Q000378:metabolism*; D014162:Transfection / Q000379:methods
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D018957:CD59 Antigens; D001360:Avidin; D011094:Polyethyleneimine; D004247:DNA