Receptor-mediated endocytosis of transferrin-polycation conjugates: an efficient way to introduce DNA into hematopoietic cells.
Most current gene transfer methods function satisfactorily in specialized systems involving established cell lines but are often not applicable with nonadherent, primary hematopoietic cells, which are notoriously difficult to transfect. To approach this problem, we have investigated an alternative method of gene transfer, "transferrinfection," in which DNA complexed to transferrin-polycation conjugates is introduced into cells by receptor-mediated endocytosis [Wagner, E., Zenke, M., Cotten, M., Beug, H. & Birnstiel, M. L. (1990) Proc. Natl. Acad. Sci. USA 87, 3410-3414]. We show here that transferrin-polylysine and transferrin-protamine, when complexed to plasmid DNA containing a luciferase reporter gene, is efficiently bound and moved into avian erythroblasts by endocytosis. Successful transfer and expression of the luciferase reporter gene depends on specific interaction of the transferrin-polylysine-DNA complex with the transferrin receptor and occurs in a significant fraction (greater than 95%) of the cells. Gene transfer efficiency by transferrinfection is lower than with an optimized DEAE-dextran transfection method but reaches similar efficiencies when the cells are treated with chloroquine. Because the procedure in the absence of chloroquine is completely nontoxic to cells, a constant expression level of transferred genes may be maintained by repeated additions of transferrin-polylysine-DNA complex. In addition, the usefulness of transferrinfection for gene transfer into primary hematopoietic cells is demonstrated.
Zenke
M
M
Research Institute of Molecular Pathology, Vienna, Austria.
Steinlein
P
P
Wagner
E
E
Cotten
M
M
Beug
H
H
Birnstiel
M L
ML
D000818:Animals; D002412:Cations; D002645:Chickens; D002738:Chloroquine / Q000494:pharmacology; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D004705:Endocytosis; D004900:Erythroblasts / Q000187:drug effects / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D007700:Kinetics; D010957:Plasmids; D011107:Polylysine / Q000378:metabolism; D011479:Protamines / Q000378:metabolism; D011485:Protein Binding; D011990:Receptors, Transferrin / Q000378:metabolism*; D014162:Transfection; D014168:Transferrin / Q000031:analogs & derivatives* / Q000378:metabolism*
D002412:Cations; D011479:Protamines; D011990:Receptors, Transferrin; D014168:Transferrin; D011107:Polylysine; D002738:Chloroquine; D004247:DNA