Targeted gene modification in mismatch-repair-deficient embryonic stem cells by single-stranded DNA oligonucleotides.
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.
Dekker
Marleen
M
Division of Molecular Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
Brouwers
Conny
C
te Riele
Hein
H
D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D004260:DNA Repair / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000235:genetics; D004268:DNA-Binding Proteins; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism*; D016368:Frameshift Mutation / Q000235:genetics; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D051379:Mice; D051718:MutS Homolog 2 Protein; D009154:Mutation; D009841:Oligonucleotides / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000172:deficiency / Q000235:genetics; D016160:Retinoblastoma Protein / Q000235:genetics; D017384:Sequence Deletion; D013234:Stem Cells / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D011518:Proto-Oncogene Proteins; D016160:Retinoblastoma Protein; C497173:Msh2 protein, mouse; D051718:MutS Homolog 2 Protein