Preparation of plasmid DNA in transfection complexes for fluorescence and electron spectroscopic imaging.
The aim of this project was to develop procedures necessary to study mechanisms of receptor mediated gene transfer by means of integrated microscopy. Plasmid DNA was incorporated into a transfection complex consisting of poly(L)lysine and transferrin to which the nuclear localization signal was conjugated. This complex was presented to cultured glioma cells. Preparation of the transfected DNA for imaging was pursued by two methods. In the first method tetramethylrhodamine, nanogold, and ferritin were linked through streptavidin to the biotinylated plasmid DNA. Trafficking of the fluorescent derivatives was studied in living cells with fluorescence microscopy. Then, selected cells were rapidly cryo-immobilized. Ultra-structural distribution of the transfected DNA was imaged with energy filtering transmission electron microscopy. In the second method, the unmodified transfected DNA was detected in cryo-immobilized cells by in situ polymerase chain reaction and in situ hybridization. For laser scanning fluorescence microscopy probes were labeled with tetramethylrhodamine. For ultrastructural analysis by electron spectroscopic imaging, probes containing incorporated digoxigenin were labeled with anti-digoxigenin boronated antibodies. Based upon the developed procedures, it has been demonstrated that the presence of the nuclear localization signal in the transfection complex resulted in rapid nuclear import of the transfected DNA.
Malecki
M
M
Molecular Biology Laboratory, University of Wisconsin at Madison 53706, USA. malecki@macc.wisc.edu
D015925:Cryopreservation; D004247:DNA / Q000032:analysis*; D018014:Gene Transfer Techniques; D016591:Histocytological Preparation Techniques; D006801:Humans; D017403:In Situ Hybridization; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000235:genetics / Q000648:ultrastructure*; D016133:Polymerase Chain Reaction; D014162:Transfection; D014407:Tumor Cells, Cultured
D004247:DNA