GLYCEROL AND POLYLYSINE SYNERGIZE IN THEIR ABILITY TO RUPTURE VESICULAR MEMBRANES - A MECHANISM FOR INCREASED TRANSFERRIN-POLYLYSINE-MEDIATED GENE-TRANSFER

The presence of about 1.2 M glycerol during transfection with DNA/tran sferrin-polylysine and DNA/polylysine complexes dramatically increases transgene expression in a variety of cell types, provided that the co mplexes have an excess of polylysine. We have characterized this pheno menon using a human melanoma cell line (H225), The addition of 1.2 M g lycerol to the transfection medium has no influence on the internaliza tion of DNA complexes or on the promoter activity used to direct repor ter gene expression. Neither prenor postincubation of the cells with g lycerol results in a notable increase in transgene expression. Bafilom ycin Al and chloroquine, two drugs affecting the endosomal pathway, bo th influenced transgene expression, indicating that glycerol acts on i nternal vesicles. Glycerol and polylysine synergized in their ability to lyse erythrocytes as well as internal vesicles (microsomes) isolate d from H225 cells, indicating that the glycerol effect is due to a lab ilization of vesicular membranes, which facilitates membrane disruptio n by polylysine. Our current model suggests that the excess of polylys ine in the DNA complexes disrupts vesicular membranes in the presence of glycerol, thus allowing the release of DNA complexes into the cytop lasm. (C) 1997 Academic Press.