Targeted gene modification in mismatch‐repair‐deficient embryonic stem cells by single‐stranded DNA oligonucleotides
M Dekker, C Brouwers, H te Riele - Nucleic Acids Research, 2003 - academic.oup.com
M Dekker, C Brouwers, H te Riele
Nucleic Acids Research, 2003•academic.oup.comGene 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 …
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 …
Abstract
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.
Oxford University Press