Branched oligonucleotides induce in vivo gene conversion of a mutated EGFP reporter
- 8 September 2003
- journal article
- Published by Springer Nature in Gene Therapy
- Vol. 10 (21) , 1830-1840
- https://doi.org/10.1038/sj.gt.3302079
Abstract
Branched oligonucleotides (b-oligonucleotides) based on a novel branching monomer were used for site-specific sequence alteration in vivo. With a stable integrated mutated enhanced green fluorescent protein (EGFP) template in Chinese hamster ovary cells, up to 0.1% EGFP-positive cells were counted after transfection with b-oligonucleotides. The presence of EGFP protein in converted cells was demonstrated by anti-EGFP immunocytochemistry. Genomic sequencing of converted cells showed in 40% of the analysed clones the corrected wild-type codon, while 9.3% of the sequences showed a corrected wild-type sequence and an additional collateral mutation. Despite the stable corrected genomic locus, converted cells entered selective apoptosis after 3–6 days. The cell line Irs-1 that is deficient in the homologous recombination pathway showed a reduced frequency of b-oligonucleotide-induced site-specific sequence conversion. The reduced conversion rates in the mutant cell line could be partly rescued by complementation with XRCC2 cDNA.Keywords
This publication has 39 references indexed in Scilit:
- Single-stranded DNA Induces Ataxia Telangiectasia Mutant (ATM)/p53-dependent DNA Damage and Apoptotic SignalsJournal of Biological Chemistry, 2003
- Optimising gene repair strategies in cell cultureGene Therapy, 2002
- Functional correction of episomal mutations withshort DNA fragments and RNA–DNA oligonucleotidesThe Journal of Gene Medicine, 2002
- Phosphodiester Oligonucleotides Inhibit Mitosis and Trigger Apoptosis by a Non-Antisense, p53-Mediated MechanismAntisense and Nucleic Acid Drug Development, 2002
- XRCC2 Is a Nuclear RAD51-like Protein Required for Damage-dependent RAD51 Focus Formation without the Need for ATP BindingPublished by Elsevier ,2001
- Expression of ΔF508 CFTR in normal mouse lung after site-specific modification of CFTR sequences by SFHRGene Therapy, 2001
- Triplex-induced Recombination in Human Cell-free ExtractsPublished by Elsevier ,2001
- Homologous Recombination Based Gene TherapyNephron Experimental Nephrology, 1999
- Triplex‐Mediated, in vitro Targeting of Psoralen Photoadducts within the Genome of a Transgenic MousePhotochemistry and Photobiology, 1996
- Mutant rodent cell lines sensitive to ultraviolet light, ionizing radiation and cross-linking agents: a comprehensive survey of genetic and biochemical characteristicsMutation Research/DNA Repair, 1993