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- Title
Branched oligonucleotides induce in vivo gene conversion of a mutated EGFP reporter.
- Authors
Olsen, P.A.; McKeen, C.; Krauss, S.
- 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.Gene Therapy (2003) 10, 1830-1840. doi:10.1038/sj.gt.3302079
- Subjects
OLIGONUCLEOTIDES; GREEN fluorescent protein; IMMUNOCYTOCHEMISTRY; GENE therapy
- Publication
Gene Therapy, 2003, Vol 10, Issue 21, p1830
- ISSN
0969-7128
- Publication type
Article
- DOI
10.1038/sj.gt.3302079