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 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.
El Ouahabi
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A
Laboratoire de Chimie-Physique des Macromolécules aux Interfaces (LCPMI), Université Libre de Bruxelles, Brussels.
Thiry
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M
Schiffmann
S
S
Fuks
R
R
Nguyen-Tran
H
H
Ruysschaert
J M
JM
Vandenbranden
M
M
D000578:Amidines / Q000378:metabolism*; D000818:Animals; D001973:Bromodeoxyuridine / Q000032:analysis; D002460:Cell Line; D006224:Cricetinae; D004705:Endocytosis; D007150:Immunohistochemistry; D008561:Membrane Fusion; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000378:metabolism*
D000578:Amidines; C089665:N-t-butyl-n'-tetradecyl-3-tetradecylaminopropionamidine; D001973:Bromodeoxyuridine