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

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Version: Final published version
Serval ID
serval:BIB_0F962F3951C9
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
Stable transmission of targeted gene modification using single-stranded oligonucleotides with flanking LNAs.
Journal
Nucleic Acids Research
Author(s)
Andrieu-Soler C., Casas M., Faussat A.M., Gandolphe C., Doat M., Tempé D., Giovannangeli C., Behar-Cohen F., Concordet J.P.
ISSN
1362-4962 (Electronic)
ISSN-L
0305-1048
Publication state
Published
Issued date
2005
Peer-reviewed
Oui
Volume
33
Number
12
Pages
3733-3742
Language
english
Notes
Publication types: Comparative Study ; Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, P.H.S.Publication Status: epublish
Abstract
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 betaPDE 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.
Keywords
3',5'-Cyclic-GMP Phosphodiesterases/genetics, Animals, Cell Line, Cell Survival, Chromosomes, Mammalian, Cyclic Nucleotide Phosphodiesterases, Type 6, DNA, Single-Stranded/chemistry, Green Fluorescent Proteins/genetics, Luminescent Proteins/genetics, Mice, Mutagenesis, Mutation, Oligonucleotides, Oligonucleotides, Antisense/chemistry, Photoreceptor Cells, Vertebrate/cytology, Plasmids, Sequence Analysis, DNA, Transgenes
Pubmed
Web of science
Open Access
Yes
Create date
08/11/2013 11:16
Last modification date
20/08/2019 12:36
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