Receptor-mediated in vitro gene transformation by a soluble DNA carrier system.
We present, here, evidence that foreign DNA can be specifically delivered to cells by a soluble carrier system that takes advantage of receptor-mediated endocytosis. Our experiments were based on the following concepts: hepatocytes possess a unique receptor that binds and internalizes galactose-terminal (asialo-)glycoproteins; DNA can bind to polycations in a strong but noncovalent manner forming soluble complexes; and the gene for chloramphenicol acetyltransferase, a bacterial enzyme that acetylates chloramphenicol, is not present in mammalian cells. We coupled asialoorosomucoid (ASOR) to poly-L-lysine to form an asialoorosomucoid-poly-L-lysine conjugate. The plasmid, pSV2 CAT, was complexed to the conjugate in a molar ratio of 1:2. To test this complex, a model system was used consisting of hepatoma cell lines, Hep G2, asialoglycoprotein receptor (+), and SK-Hep 1, receptor (-). Each cell line was incubated with filtered ASOR X poly-L-lysine X DNA complex, or controls consisting of DNA plus ASOR, DNA plus poly-L-lysine, or DNA alone. Cells were assayed for the presence of chloramphenicol acetyltransferase activity as a measure of gene transformation. SK-Hep 1, receptor (-) cells, produced no detectable acetylated chloramphenicol derivatives under any condition. However, Hep G2, receptor (+) cells, incubated with the ASOR X poly-L-lysine X DNA complex were transformed as indicated by the presence of chloramphenicol acetyltransferase activity (0.028 chloramphenicol acetyltransferase units/10(6) cells). Mixtures of individual components of the complex failed to transform these cells. Competition by a 10-fold excess of ASOR prevented gene transformation by the ASOR X poly-L-lysine X DNA complex.
D000818:Animals; D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; D001693:Biological Transport, Active; D002460:Cell Line; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D008114:Liver Neoplasms, Experimental / Q000235:genetics / Q000378:metabolism; D009961:Orosomucoid / Q000031:analogs & derivatives / Q000378:metabolism; D010957:Plasmids; D011107:Polylysine / Q000378:metabolism; D011971:Receptors, Immunologic / Q000378:metabolism*; D012995:Solubility; D014170:Transformation, Genetic
D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; D009961:Orosomucoid; D011971:Receptors, Immunologic; C017030:asialoorosomucoid; D011107:Polylysine; D004247:DNA