Intranuclear fluorescence resonance energy transfer analysis of plasmid DNA decondensation from nonviral gene carriers.

BACKGROUND

There has been considerable interest in researching the regulatory mechanisms that control transgene expression. In particular, there is impetus to investigate the intranuclear mechanisms for gene expression in order to improve the transfection efficiency of nonviral gene carriers.

METHODS

To clarify the direct relationship between DNA decondensation and gene expression, plasmid DNA encoding Keima-Red fluorescent protein was doubly-labeled by a pair of donor-acceptor fluorescent dyes (fluorescein and Cy3), and transfected to HuH-7 cells using nonviral gene carriers: polyethylenimine (polyplex) and LipofectAMINE 2000 (lipoplex). Fluorescence resonance energy transfer analysis between the two dyes represents the condensation state of the pDNA. The intranuclear trafficking and condensation state of the pDNA were explored in transgene expressing and non-expressing cells under confocal laser scanning spectromicroscopy.

RESULTS

The majority of transgene positively expressing cells transfected with polyplex and lipoplex had decondensed pixels in the nucleus. The majority of non-expressing cells transfected with polyplex had only condensed pixels in the nucleus. The majority of non-expressing cells transfected with lipoplex had no pixels in the nucleus.

CONCLUSIONS

Both polyplex and lipoplex marked a strong correlation between pDNA decondensation and transgene expression, yet the major limiting factor for transgene expression was different. In polyplex, the pDNA decondensation after nuclear entry was the major determining factor for transgene expression, whereas the nuclear entry itself was the chief determining step for transgene expression by lipoplex. This imaging technique allowed in situ observation of pDNA in the nucleus, providing important information about DNA behavior for gene expression.