Stable transmission of targeted gene modification using single-stranded oligonucleotides with flanking LNAs.
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.
INSERM U598, Institut Biomédical des Cordeliers 15 rue de l'Ecole de Médecine, 75270 Paris Cedex 06, France.
Charlotte
C
Andrieu-Soler
Mariana
M
Casas
Anne-Marie
AM
Faussat
Christelle
C
Gandolphe
Marc
M
Doat
Denis
D
Tempé
Carine
C
Giovannangeli
Francine
F
Behar-Cohen
Jean-Paul
JP
Concordet
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000235:genetics; D000818:Animals; D002460:Cell Line; D002470:Cell Survival; D033481:Chromosomes, Mammalian; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D004277:DNA, Single-Stranded / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D016296:Mutagenesis; D009154:Mutation; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense / Q000737:chemistry*; D020419:Photoreceptor Cells, Vertebrate / Q000166:cytology; D010957:Plasmids; D017422:Sequence Analysis, DNA; D019076:Transgenes
D004277:DNA, Single-Stranded; D008164:Luminescent Proteins; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; C409504:blue fluorescent protein, Aequorea victoria; C477371:locked nucleic acid; D049452:Green Fluorescent Proteins; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; C517438:Pde6b protein, mouse