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Effective oligonucleotide-mediated gene disruption in ES cells lacking the mismatch repair protein MSH3

Gene Therapy volume 13, pages 686–694 (2006)Cite this article

Abstract

We have previously demonstrated that site-specific insertion, deletion or substitution of one or two nucleotides in mouse embryonic stem cells (ES cells) by single-stranded deoxyribo-oligonucleotides is several hundred-fold suppressed by DNA mismatch repair (MMR) activity. Here, we have investigated whether compound mismatches and larger insertions escape detection by the MMR machinery and can be effectively introduced in MMR-proficient cells. We identified several compound mismatches that escaped detection by the MMR machinery to some extent, but could not define general rules predicting the efficacy of complex base-pair substitutions. In contrast, we found that four-nucleotide insertions were largely subject to suppression by the MSH2/MSH3 branch of MMR and could be effectively introduced in Msh3-deficient cells. As these cells have no overt mutator phenotype and Msh3-deficient mice do not develop cancer, Msh3-deficient ES cells can be used for oligonucleotide-mediated gene disruption. As an example, we present disruption of the Fanconi anemia gene Fancf.

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Acknowledgements

We thank Floris Foijer and Eva Wielders for valuable comments on the manuscript and Guus Hart for help with statistical analyses. This work was supported by grants from the Dutch Cancer Society (NKI 2000-2233) and the Netherlands Genomics Initiative (Horizon 050-71-007).

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Authors and Affiliations

  1. Division of Molecular Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands

    M Dekker, C Brouwers, M Aarts, J van der Torre, S de Vries & H te Riele

  2. Department of Clinical Genetics, Free University Medical Centre, Amsterdam, The Netherlands

    H van de Vrugt

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  1. M Dekker
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  2. C Brouwers
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  4. J van der Torre
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Correspondence to H te Riele.

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Dekker, M., Brouwers, C., Aarts, M. et al. Effective oligonucleotide-mediated gene disruption in ES cells lacking the mismatch repair protein MSH3. Gene Ther 13, 686–694 (2006). https://doi.org/10.1038/sj.gt.3302689

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