title: Transient suppression of MLH1 allows effective single-nucleotide substitution by single-stranded DNA oligonucleotides.
abstract: Short synthetic single-stranded oligodeoxyribonucleotides (ssODNs) can be used to introduce subtle modifications into the genome of mouse embryonic stem cells (ESCs). We have previously shown that effective application of ssODN-mediated gene targeting in ESC requires (transient) suppression of DNA mismatch repair (MMR). However, whereas transient down-regulation of the mismatch recognition protein MSH2 allowed substitution of 3 or 4 nucleotides, 1 or 2 nucleotide substitutions were still suppressed. We now demonstrate that single- or dinucleotide substitution can effectively be achieved by transient down-regulation of the downstream MMR protein MLH1. By exploiting highly specific real-time PCR, we demonstrate the feasibility of substituting a single basepair in a non-selectable gene. However, disabling the MMR machinery may lead to inadvertent mutations. To obtain insight into the mutation rate associated with transient MMR suppression, we have compared the impact of transient and constitutive MMR deficiency on the repair of frameshift intermediates at mono- and dinucleotide repeats. Repair at these repeats relied on the substrate specificity and functional redundancy of the MSH2/MSH6 and MSH2/MSH3 MMR complexes. MLH1 knockdown increased the level of spontaneous mutagenesis, but modified ESCs remained germ line competent. Thus, transient MLH1 suppression provides a valuable extension of the MSH2 knockdown strategy, allowing rapid generation of mice carrying single basepair alterations in their genome.
authors.affiliation: Division of Molecular Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
authors.forename: Marleen
authors.initials: M
authors.lastname: Dekker
authors.forename: Sandra
authors.initials: S
authors.lastname: de Vries
authors.forename: Marieke
authors.initials: M
authors.lastname: Aarts
authors.forename: Robert
authors.initials: R
authors.lastname: Dekker
authors.forename: Conny
authors.initials: C
authors.lastname: Brouwers
authors.forename: Oliver
authors.initials: O
authors.lastname: Wiebenga
authors.forename: Niels
authors.initials: N
authors.lastname: de Wind
authors.forename: Erika
authors.initials: E
authors.lastname: Cantelli
authors.forename: Roberto
authors.initials: R
authors.lastname: Tonelli
authors.forename: Hein
authors.initials: H
authors.lastname: Te Riele
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D053843:DNA Mismatch Repair; D004277:DNA, Single-Stranded; D018900:Dinucleotide Repeats; D015536:Down-Regulation; D053595:Embryonic Stem Cells; D016368:Frameshift Mutation; D055785:Gene Knockdown Techniques; D018390:Gene Targeting; D051379:Mice; D000070957:MutL Protein Homolog 1; D016296:Mutagenesis; D009687:Nuclear Proteins / Q000378:metabolism*; D009841:Oligonucleotides
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D004277:DNA, Single-Stranded; C501485:Mlh1 protein, mouse; D009687:Nuclear Proteins; D009841:Oligonucleotides; D000070957:MutL Protein Homolog 1