Biochemical and Biophysical Research Communications
Visualization of intracellular trafficking of exogenous DNA delivered by cationic liposomes
Section snippets
Materials and methods
General. NIH3T3 cells were from the American Type Culture Collection (Manassas, Virginia). COS-7 cells were from the RIKEN Cell Bank (Tsukuba, Japan). SYTO 24 was purchased from Molecular Probes (Eugene, Oregon). Calf thymus histones (mixture of H1, H2A, H2B, H3, and H4) were from Roche Diagnostics.
Transfection. The pQBI25 plasmid (5 μg; Takara, Shiga, Japan) was labeled with Rh by the Label IT Rhodamine Labeling Kit (PanVera, Madison, Wisconsin) and purified by ethanol precipitation. DNA
Time course of transfection
We observed NIH3T3 cells transfected with Rh-labeled DNA (observed in red) by confocal microscopy. SYTO 24 was used to label the nucleus; this reagent labels DNA fluorescently (in green).
Surprisingly, some cells contained the labeled DNAs in their nuclei at 30 min (Fig. 1A). After a 1-h incubation, similar images were obtained by confocal microscopy (Fig. 1B). In this case, two of the four cells contained the DNAs in their nuclei. Collectively, in 14 of the 36 cells analyzed, the labeled DNAs
Discussion
By the use of fluorescently labeled DNA and confocal microscopy, we observed several interesting features of intracellular trafficking of exogenous DNA transfected with cationic liposomes. First, the DNA was able to enter the nucleus within a very short period of time (Figs. 1A–C). One speculated mechanism for the nuclear entry is that the DNA enters when the nuclear membrane disappears at the M stage of cell division. Since the doubling time of NIH3T3 cells under our conditions was about 24 h,
Acknowledgements
This work was supported in part by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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