Improving gene replacement by intracellular formation of linear homologous DNA
Article first published online: 10 JAN 2005
Copyright © 2005 John Wiley & Sons, Ltd.
The Journal of Gene Medicine
Volume 7, Issue 5, pages 649–656, May 2005
How to Cite
de Piédoue, G., Maurisse, R., Kuzniak, I., Lopez, B., Perrin, A., Nègre, O., Leboulch, P. and Feugeas, J.-P. (2005), Improving gene replacement by intracellular formation of linear homologous DNA. J. Gene Med., 7: 649–656. doi: 10.1002/jgm.706
- Issue published online: 3 MAY 2005
- Article first published online: 10 JAN 2005
- Manuscript Accepted: 24 SEP 2004
- Manuscript Revised: 17 SEP 2004
- Manuscript Received: 21 MAY 2004
- INSERM (Institut National de la Santé et de la Recherche Médicale), AFM (Association Française contre les Myopathies), ARC (Association pour la Recherche contre le Cancer) and Ligue contre le cancer
- gene targeting;
- homologous recombination
Gene targeting is a potential tool for gene therapy but is limited by the low rate of homologous recombination. Using highly homologous linear DNA improves gene targeting frequency but requires microinjection into nuclear cells to be effective. Because transfection of circular DNA is more efficient than transfection of linear DNA and adaptable to viral vectors, we developed a system for the intracellular release of linear fragments from circular plasmids.
Only one cutting site inside the “donor” DNA was not convenient because it led to integration of exogenous sequences into the target. So we constructed several “donor” plasmids containing the homologous sequences flanked by two I-Sce I recognition sites. Expression of I-Sce I allowed intracellular delivery of “ends-out” (replacement) vectors. We compared the efficiency of different constructions to correct a mutated gfp target.
Co-transfection of “donor” plasmids and an I-Sce I expression vector into CHO cells enhanced the correction of an extrachromosomal mutated gfp target by at least 10 times. Maximum correction was observed with the greatest homology size and maximum effect of I-Sce I was obtained when the long hemi-sites of the duplicated I-Sce I sites were contiguous to the homologous sequence. Unexpectedly, the reverse orientation of I-Sce I sites provided little or no effect, probably due to the asymmetrical activity of the I-Sce I meganuclease.
Releasing homologous DNA fragments with I-Sce I enhances gene replacement. This work provides the basis for the future design of viral vectors for gene replacement. Copyright © 2005 John Wiley & Sons, Ltd.