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Journal of Histochemistry and Cytochemistry, Vol. 47, 1159-1166, September 1999, Copyright © 1999, The Histochemical Society, Inc.

ARTICLE

Intracellular Visualization of BrdU-labeled Plasmid DNA/Cationic Liposome Complexes

Abdelatif El Ouahabia, Marc Thiryb, Serge Schiffmannc, Robert Fuksd, Huy Nguyen-Tranc, Jean-Marie Ruysschaerta, and Michel Vandenbrandena
a Laboratoire de Chimie-Physique des Macromolécules aux Interfaces (LCPMI), Université Libre de Bruxelles, Brussels
b Laboratoire de Biologie Cellulaire et Tissulaire, Université de Liège, Liège
c Laboratoire de Neuropathologie et de Recherche sur les Neuropeptides, Université Libre de Bruxelles, Brussels
d Laboratoire de Chimie Générale, Institut de Pharmacie, Université Libre de Bruxelles, Brussels, Belgium

Correspondence to: Abdelatif El Ouahabi, Laboratorie de Chemie–Physique des Macromolécules aux Interfaces, Université Libre de Bruxelles, Campus Plaine CP 206/2, B-1050 Brussels, Belgium.

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 lipid, Vectamidine (3-tetradecylamino-N-tert-butyl-N'-tetradecylpropionamidine) in BHK21 cells. To facilitate its detection inside the cells, bromodeoxyuridine (BrdU) was incorporated into plasmid DNA under conditions that minimize plasmid alteration. BrdU was localized in cells incubated with Vectamidine/BrdU-labeled plasmid DNA complexes by immunogold labeling and electron microscopy (EM). Labeling was predominantly associated with aggregated liposome structures at the surface of and inside the cells. EM observations of cells transfected with Vectamidine/DNA complexes showed that the liposome/DNA aggregates accumulate in large vesicles in the cell cytosol. On the other hand, using rhodamine-labeled Vectamidine and revealing BrdU with FITC-conjugated antibodies permitted simultaneous detection in the cells of both components 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 BrdU-labeled plasmid DNA detection system can be a useful tool to visualize exogenous DNA entry into cells by a combination of electron and confocal microscopy. (J Histochem Cytochem 47:1159–1166, 1999)

Key Words: transfection, plasmid DNA, BrdU, cationic liposome, confocal laser scanning microscopy, electron microscopy


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