Intracellular visualization of BrdU-labeled plasmid DNA/cationic liposome complexes

Difficulties in specific detection of transfected DNA in cells represent an important limitation in the study of the gene transfer process. We studied the cellular entry and fate of a plasmid DNA complexed with a cationic lip id, Vectamidine (3-tetradecylamino-N-tert-butyl-N'-tetradecylpropionamidine ) in BHK21 cells. To facilitate its detection inside the cells, bromodeoxyu ridine (BrdU) was incorporated into plasmid DNA under conditions that minim ize plasmid alteration. BrdU was localized in cells incubated with Vectamid ine/BrdU-labeled plasmid DNA complexes by immunogold labeling and electron microscopy (EM). Labeling was predominantly associated with aggregated lipo some structures at the surface of and inside the cells. EM observations of cells transfected with Vectamidine/DNA complexes showed that the liposome/D NA aggregates accumulate in large vesicles in the cell cytosol. On the othe r hand, using rhodamine-labeled Vectamidine and revealing BrdU with FITC-co njugated antibodies permitted simultaneous detection in the cells of both c omponents of the complexes with confocal laser scanning microscopy. The DNA and lipids co-localized at the surface of and inside the cells, indicating that the complex is internalized as a whole. Our results show that the Brd U-labeled plasmid DNA detection system can be a useful tool to visualize ex ogenous DNA entry into cells by a combination of electron and confocal micr oscopy.