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Open Access Highly Accessed Methodology article

MAR-mediated integration of plasmid vectors for in vivo gene transfer and regulation

Stefania Puttini1, Ruthger W van Zwieten1, Damien Saugy1, Małgorzata Lekka2, Florence Hogger1, Deborah Ley1, Andrzej J Kulik3 and Nicolas Mermod14*

Author Affiliations

1 Institute of Biotechnology, University of Lausanne, Lausanne, Switzerland

2 The Henryk Niewodniczański Institute of Nuclear Physics, Polish Academy of Sciences, Kraków, Poland

3 Laboratory of Physics of Living Matter - IPSB, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland

4 Laboratory for Molecular Biotechnology, Station 6, EPFL, 1015 Lausanne, Switzerland

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BMC Molecular Biology 2013, 14:26  doi:10.1186/1471-2199-14-26

Published: 2 December 2013

Abstract

Background

The in vivo transfer of naked plasmid DNA into organs such as muscles is commonly used to assess the expression of prophylactic or therapeutic genes in animal disease models.

Results

In this study, we devised vectors allowing a tight regulation of transgene expression in mice from such non-viral vectors using a doxycycline-controlled network of activator and repressor proteins. Using these vectors, we demonstrate proper physiological response as consequence of the induced expression of two therapeutically relevant proteins, namely erythropoietin and utrophin. Kinetic studies showed that the induction of transgene expression was only transient, unless epigenetic regulatory elements termed Matrix Attachment Regions, or MAR, were inserted upstream of the regulated promoters. Using episomal plasmid rescue and quantitative PCR assays, we observed that similar amounts of plasmids remained in muscles after electrotransfer with or without MAR elements, but that a significant portion had integrated into the muscle fiber chromosomes. Interestingly, the MAR elements were found to promote plasmid genomic integration but to oppose silencing effects in vivo, thereby mediating long-term expression.

Conclusions

This study thus elucidates some of the determinants of transient or sustained expression from the use of non-viral regulated vectors in vivo.

Keywords:
Inducible expression; Non-viral vectors; MAR elements; Erythropoietin; Utrophin; Gene therapy