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Nucleic Acids Research 2005 33(12):3733-3742; doi:10.1093/nar/gki686
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Published online 7 July 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oupjournals.org

Article

Stable transmission of targeted gene modification using single-stranded oligonucleotides with flanking LNAs

Charlotte Andrieu-Soler1, Mariana Casas2, Anne-Marie Faussat1, Christelle Gandolphe1, Marc Doat1, Denis Tempé2, Carine Giovannangeli3, Francine Behar-Cohen1,4 and Jean-Paul Concordet2,*

1INSERM U598, Institut Biomédical des Cordeliers 15 rue de l'Ecole de Médecine, 75270 Paris Cedex 06, France 2Département Génétique et Développement, Institut Cochin, INSERM U567, CNRS UMR8104 24 rue du Faubourg St-Jacques, 75014 Paris, France 3Laboratoire de Biophysique, Muséum National d'Histoire Naturelle, INSERM, U565, CNRS UMR 5153 7505 Paris, France 4Fondation Ophtalmologique Rothschild Paris, France

*To whom correspondence should be addressed. Tel: +33 1 44412436; Fax: +33 1 44412421; Email: concordet{at}cochin.inserm.fr

Received April 11, 2005. Revised June 14, 2005. Accepted June 14, 2005.

Targeted mutagenesis directed by oligonucleotides (ONs) is a promising method for manipulating the genome in higher eukaryotes. In this study, we have compared gene editing by different ONs on two new target sequences, the eBFP and the rd1 mutant photoreceptor ßPDE cDNAs, which were integrated as single copy transgenes at the same genomic site in 293T cells. Interestingly, antisense ONs were superior to sense ONs for one target only, showing that target sequence can by itself impart strand-bias in gene editing. The most efficient ONs were short 25 nt ONs with flanking locked nucleic acids (LNAs), a chemistry that had only been tested for targeted nucleotide mutagenesis in yeast, and 25 nt ONs with phosphorothioate linkages. We showed that LNA-modified ONs mediate dose-dependent target modification and analyzed the importance of LNA position and content. Importantly, when using ONs with flanking LNAs, targeted gene modification was stably transmitted during cell division, which allowed reliable cloning of modified cells, a feature essential for further applications in functional genomics and gene therapy. Finally, we showed that ONs with flanking LNAs aimed at correcting the rd1 stop mutation could promote survival of photoreceptors in retinas of rd1 mutant mice, suggesting that they are also active in vivo.


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