Stable transmission of targeted gene modification using single-stranded oligonucleotides with flanking LNAs
| Autor: | Jean-Paul Concordet, Denis Tempé, Christelle Gandolphe, Carine Giovannangeli, Charlotte Andrieu-Soler, M. Doat, Francine Behar-Cohen, Mariana Casas, Anne-Marie Faussat |
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| Rok vydání: | 2005 |
| Předmět: |
Cell Survival
Green Fluorescent Proteins Mutant Oligonucleotides DNA Single-Stranded Mutagenesis (molecular biology technique) Biology medicine.disease_cause Article Cell Line Mice 03 medical and health sciences chemistry.chemical_compound Genome editing 3' 5'-Cyclic-GMP Phosphodiesterases Sense (molecular biology) Genetics medicine Animals Transgenes 3' 5'-Cyclic-GMP Phosphodiesterases/genetics Chromosomes Mammalian Cyclic Nucleotide Phosphodiesterases Type 6 DNA Single-Stranded/chemistry Green Fluorescent Proteins/genetics Luminescent Proteins/genetics Mutagenesis Mutation Oligonucleotides Antisense/chemistry Photoreceptor Cells Vertebrate/cytology Plasmids Sequence Analysis DNA 030304 developmental biology 0303 health sciences Oligonucleotide 030302 biochemistry & molecular biology Oligonucleotides Antisense Cell biology Luminescent Proteins chemistry Functional genomics DNA Photoreceptor Cells Vertebrate |
| Zdroj: | Nucleic Acids Research, vol. 33, no. 12, pp. 3733-3742 Nucleic Acids Research |
| ISSN: | 1362-4962 0305-1048 |
| DOI: | 10.1093/nar/gki686 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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