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 mechan isms 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 conj ugated. This complex was presented to cultured glioma cells. Preparation of the transfected DNA for imaging was pursued by two methods. In the first m ethod tetramethylrhodamine, nanogold, and ferritin were linked through stre ptavidin to the biotinylated plasmid DNA. Trafficking of the fluorescent de rivatives was studied in living cells with fluorescence microscopy. Then, s elected cells were rapidly cryo-immobilized. Ultrastructural distribution o f the transfected DNA was imaged with energy filtering transmission electro n microscopy. In the second method, the unmodified transfected DNA was dete cted 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 lab eled with anti-digoxigenin boronated antibodies. Based upon the developed procedures, it has been demonstrated that the pres ence of the nuclear localization signal in the transfection complex resulte d in rapid nuclear import of the transfected DNA.