David Curiel

Transferrinfection: a highly efficient way to express gene constructs in eukaryotic cells.

The expression of antisense RNA is a powerful means of extinguishing unwanted gene activity in the eukaryotic cell (see this volume). This approach has worked particularly well in plantst4 and animals5 in which antisense-producing gene constructs can be inserted into the germ cell. In man, germ cell manipulation is not yet possible. The problem arises as to how somatic cells can be targeted with genes generating antisense RNAs. Apart from retroviral vectors, a multitude of techniques exist for introducing DNA into eukaryotic somatic cells (ref. 6 and references therein). During the last few years, we and others7J have been interested in adapting the cellular mechanisms of receptor-mediated endocytosis of macromolecules for the importation of DNA molecules into cell^.^-^* The principle of the method is to covalently connect a polycation, usually polylysine, to a ligand for a receptor on the cell surface. DNA, usually in the form of cloned genes, is then bound ionically to the polylysine. During importation of the DNA by receptor-mediated endocytosis the ligand binds to the receptor, and during internalization of both of these entities the DNA is thought to become colocalized, first in the coated pits and later in the endosome. The further fate of the DNA can be particularly well studied when reporter genes such as the firefly luciferase gene or the bacterial P-galactosidase gene, driven from a viral enhancer/promoter, are employed. It can be predicted that DNA in the endosome is directed to the lysosomal compartment, where it can be expected to be destroyed by resident nucleases. Despite this, apparently a small amount of DNA does escape destruction, so that reporter gene activity is observed in a few cells of a cell population (see below). Because the genes are expressed, it is probably safe to assume that some of the DNA has reached the cell nucleus. When transferrin is used as a ligand, the transfection procedure is called “transferrinfection.” Consistent with the postulated chain of events during transferrinfection, we find that the addition of the lysosomatropic agent chloroquine, which prevents acidification and concomitant activation of lysosomal degradative enzymes, greatly enhances expression of the chosen reporter gene.“ This enhancement can be spectacular, as for instance in the human erythroleukemic cell line K562 in which subsequent to the