High-level transgene expression by homologous recombination-mediated gene transfer
- Grandjean, Mélanie Laboratory of Molecular Biotechnology, Center for Biotechnology UNIL-EPFL, University of Lausanne, Switzerland
- Girod, Pierre-Alain Laboratory of Molecular Biotechnology, Center for Biotechnology UNIL-EPFL, University of Lausanne, Switzerland
- Calabrese, David Laboratory of Molecular Biotechnology, Center for Biotechnology UNIL-EPFL, University of Lausanne, Switzerland
- Kostyrko, Kaja Laboratory of Molecular Biotechnology, Center for Biotechnology UNIL-EPFL, University of Lausanne, Switzerland
- Wicht, Marianne Service of Medical Genetics, Center Hospitalier Universitaire Vaudois (CHUV), Lausanne, Switzerland
- Yerly, Florence Department of Mathematics, University of Fribourg, Switzerland
- Mazza, Christian Department of Mathematics, University of Fribourg, Switzerland
- Beckmann, Jacques S. Service of Medical Genetics, Center Hospitalier Universitaire Vaudois (CHUV), Lausanne, Switzerland
- Martinet, Danielle Service of Medical Genetics, Center Hospitalier Universitaire Vaudois (CHUV), Lausanne, Switzerland
- Mermod, Nicolas Laboratory of Molecular Biotechnology, Center for Biotechnology UNIL-EPFL, University of Lausanne, Switzerland
-
07.06.2011
Published in:
- Nucleic Acid Research. - 2011, vol. 39, no. 15, p. e104
English
Gene transfer and expression in eukaryotes is often limited by a number of stably maintained gene copies and by epigenetic silencing effects. Silencing may be limited by the use of epigenetic regulatory sequences such as matrix attachment regions (MAR). Here, we show that successive transfections of MAR-containing vectors allow a synergistic increase of transgene expression. This finding is partly explained by an increased entry into the cell nuclei and genomic integration of the DNA, an effect that requires both the MAR element and iterative transfections. Fluorescence in situ hybridization analysis often showed single integration events, indicating that DNAs introduced in successive transfections could recombine. High expression was also linked to the cell division cycle, so that nuclear transport of the DNA occurs when homologous recombination is most active. Use of cells deficient in either non-homologous end-joining or homologous recombination suggested that efficient integration and expression may require homologous recombination-based genomic integration of MAR-containing plasmids and the lack of epigenetic silencing events associated with tandem gene copies. We conclude that MAR elements may promote homologous recombination, and that cells and vectors can be engineered to take advantage of this property to mediate highly efficient gene transfer and expression.
- Faculty
- Faculté des sciences et de médecine
- Department
- Département de Mathématiques
- Language
-
- English
- Classification
- Mathematics
- License
- License undefined
- Identifiers
-
- RERO DOC 27108
- DOI 10.1093/nar/gkr436
- Persistent URL
- https://folia.unifr.ch/unifr/documents/301992
Statistics
Document views: 41
File downloads:
- gkr436.pdf: 40