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Nucleic Acids Research, 2003, Vol. 31, No. 6 e27
© 2003 Oxford University Press

Targeted gene modification in mismatch-repair-deficient embryonic stem cells by single-stranded DNA oligonucleotides

Marleen Dekker, Conny Brouwers and Hein te Riele

Division of Molecular Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands

*To whom correspondence should be addressed. Tel: +31 20 512 20 84; Fax: +31 20 669 13 83; Email: h.t.riele{at}nki.nl

Gene targeting through homologous recombination in murine embryonic stem (ES) cells is already strongly suppressed by DNA mismatch-repair (MMR)-dependent anti-recombination when targeting construct and target locus differ at <1% of the nucleotide positions. We demonstrate that MMR activity also raises a strong impediment to gene modification mediated by small synthetic DNA oligonucleotide sequences. In the absence of the DNA MMR gene MSH2, synthetic single-stranded deoxyribo-oligonucleotides can be used to site-specifically modify the ES cell genome. We show that PCR-based procedures can be used to identify and clone modified cells. By this method we have substituted a single codon in the retinoblastoma gene.


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