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Nucleic Acids Research Advance Access published online on May 30, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn287
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Nuclear delivery of NF{kappa}B-assisted DNA/polymer complexes: plasmid DNA quantitation by confocal laser scanning microscopy and evidence of nuclear polyplexes by FRET imaging

Gilles Breuzard1, Magdalena Tertil2, Cristine Gonçalves1, Hervé Cheradame3, Philippe Géguan3, Chantal Pichon1 and Patrick Midoux1,*

1Centre de Biophysique Moléculaire CNRS UPR 4301, University of Orléans and INSERM, rue Charles Sadron, F-45071 Orléans Cedex 2, France, 2Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kzakow, Poland and 3Laboratoire Matériaux Polymères aux Interfaces CNRS UMR 7581, Université d’Evry, F-91025 Evry, France

*To whom correspondence should be addressed. Tel: +33(0) 2 3825 5595; Fax: +33(0) 2 3863 1517; Email: patrickmidoux{at}cnrs-orleans.fr

Received February 22, 2008. Revised April 25, 2008. Accepted April 25, 2008.

Quantification of a plasmid DNA (pDNA) and investigation of its polymer-associated state in the nucleus are crucial to evaluate the effectiveness of a gene-delivery system. This study was conducted with p3NF-luc-3NF, a pDNA-bearing optimized {kappa}B motif to favour NF{kappa}B-driven nuclear import. Here, a quantification of pDNA copies in the nucleus was performed by real-time confocal laser scanning microscopy in HeLa and C2C12 cells transfected with linear polyethylenimine or histidylated polylysine. Förster Resonance Energy Transfer (FRET) from the fluorescein-p3NF-luc-3NF donor to the co-localized rhodamine-polymer acceptor was carried out to investigate whether the pDNA was still condensed with the polymer in the nucleus. Upon 5 h of transfection, the nuclear amount of p3NF-luc3NF was ~1500 copies in both cell lines whereas that of pTAL-luc, a 3NF-free counterpart pDNA, was less than 250. This quantity of p3NF-luc-3NF dropped dramatically to that of pTAL-luc in the presence of the BAY 11-7085, an inhibitor of NF{kappa}B activation. These data strongly support a nuclear import of p3NF-luc3NF mediated by NF{kappa}B. Moreover, FRET experiments clearly revealed that most of nuclear pDNA were still condensed with the polymer raising the question of their passage through the nuclear pore complex and their impact on the gene-expression efficiency.

The authors wish it to be known that, in their opinion, the last two authors should be regarded as joint First Authors.


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