Gene modification in embryonic stem cells by single-stranded DNA oligonucleotides.
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.
Aarts
Marieke
M
Division of Molecular Biology, The Netherlands Cancer Institute, , Amsterdam, The Netherlands.
Dekker
Marleen
M
Dekker
Rob
R
de Vries
Sandra
S
van der Wal
Anja
A
Wielders
Eva
E
Riele
Hein Te
HT
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
D004277:DNA, Single-Stranded; C089762:Msh3 protein, mouse; D000074081:MutS Homolog 3 Protein; D009841:Oligonucleotides; D011506:Proteins