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Branched oligonucleotides induce in vivo gene conversion of a mutated EGFP reporter

Gene Therapy volume 10, pages 1830–1840 (2003)Cite this article

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.

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Acknowledgements

We thank Tom brown for his support with design and synthesis of oligonucleotides. We thank Bengt Norden, Erling Seeberg, Orlando Scharer, Keith Fox and Peter Nielsen for helpful discussions. We are especially grateful to Rolf Seljelid without whose encouragement and support the project would not have been possible. We are grateful to Gøril Olsen and Markus Randøl for excellent technical help. This research has been funded by EU Grant QLK3-CT-2000-00634, the Norwegian Research Council and the Norwegian Cancer Society.

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  1. Section for Genetic Therapy, Institute of Microbiology, The National Hospital, Oslo, Norway

    P A Olsen & S Krauss

  2. Oswel/Eurogentec, Southampton, UK

    C McKeen

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  1. P A Olsen
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  2. C McKeen
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  3. S Krauss
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Olsen, P., McKeen, C. & Krauss, S. Branched oligonucleotides induce in vivo gene conversion of a mutated EGFP reporter. Gene Ther 10, 1830–1840 (2003). https://doi.org/10.1038/sj.gt.3302079

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