title: Gene modification in embryonic stem cells by single-stranded DNA oligonucleotides.
abstract: Oligonucleotide-mediated gene targeting is an attractive alternative to current procedures to subtly modify the genome of mouse embryonic stem (ES) cells. However, oligonucleotide-directed substitution, insertion or deletion of a single or a few nucleotides was hampered by DNA mismatch repair (MMR). We have developed strategies to circumvent this problem based on findings that the central MMR protein MSH2 acts in two different mismatch recognition complexes: MSH2/MSH6, which mainly recognizes base substitutions; and MSH2/MSH3, which has more affinity for larger loops. We found that oligonucleotide-mediated base substitution could effectively be obtained upon transient suppression of MSH2 protein level, while base insertions were effective in ES cells deficient for MSH3. This method allows substitution of any codon of interest in the genome.
authors.affiliation: Division of Molecular Biology, The Netherlands Cancer Institute, , Amsterdam, The Netherlands.
authors.forename: Marieke
authors.initials: M
authors.lastname: Aarts
authors.forename: Marleen
authors.initials: M
authors.lastname: Dekker
authors.forename: Rob
authors.initials: R
authors.lastname: Dekker
authors.forename: Sandra
authors.initials: S
authors.lastname: de Vries
authors.forename: Anja
authors.initials: A
authors.lastname: van der Wal
authors.forename: Eva
authors.initials: E
authors.lastname: Wielders
authors.forename: Hein Te
authors.initials: HT
authors.lastname: Riele
mesh_terms: D000818:Animals; D001483:Base Sequence; D053843:DNA Mismatch Repair; D004277:DNA, Single-Stranded / Q000235:genetics*; D053595:Embryonic Stem Cells / Q000502:physiology*; D018390:Gene Targeting / Q000379:methods*; D051379:Mice; D008969:Molecular Sequence Data; D000074081:MutS Homolog 3 Protein; D009841:Oligonucleotides / Q000235:genetics*; D011506:Proteins / Q000502:physiology*; D012689:Sequence Homology, Nucleic Acid
chemical_list: D004277:DNA, Single-Stranded; C089762:Msh3 protein, mouse; D000074081:MutS Homolog 3 Protein; D009841:Oligonucleotides; D011506:Proteins