Visualization of intracellular trafficking of exogenous DNA delivered by cationic liposomes

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Abstract

To visualize the intracellular trafficking of exogenous DNAs delivered by cationic liposomes, rhodamine-labeled DNAs were transfected into NIH3T3 cells and observed by confocal laser microscopy. After 0.5- to 1-h incubations, the DNAs reached the nucleus with a much higher frequency than that expected from the cell division rate. This result suggests that DNAs can enter the nucleus in the presence of the nuclear membrane. Interestingly, some DNAs appeared to extend through the nuclear membrane in the aggregated form which were much larger than the nuclear pore complex. The DNAs which have passed through the nuclear membrane were stained with SYTO 24, a DNA labeling reagent. The stained part may be “naked” DNA that is free of lipids or proteins. This observation indicates that a complex containing DNA fuses with the nuclear membrane and then naked DNA is released into the nucleus.

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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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