1. Transient suppression of MLH1 allows effective single-nucleotide substitution by single-stranded DNA oligonucleotides
- Author
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Niels de Wind, Sandra S. de Vries, Erika Cantelli, Conny Brouwers, Hein te Riele, Oliver Wiebenga, Marleen Dekker, Robert Dekker, Marieke Aarts, Roberto Tonelli, M. Dekker, S. de Vrie, M. Aart, R. Dekker, C Brouwersa, O. Wiebenga, N. de Wind, E. Cantelli, R. Tonelli, and H te Riele
- Subjects
congenital, hereditary, and neonatal diseases and abnormalities ,Health, Toxicology and Mutagenesis ,Oligonucleotides ,DNA, Single-Stranded ,Down-Regulation ,Biology ,DNA Mismatch Repair ,Frameshift mutation ,Cell Line ,Mice ,Genetics ,Gene Knockdown Techniques ,Animals ,Dinucleotide Repeats ,Frameshift Mutation ,Molecular Biology ,Embryonic Stem Cells ,Adaptor Proteins, Signal Transducing ,Oligonucleotide ,Mutagenesis ,Gene targeting ,Nuclear Proteins ,Molecular biology ,digestive system diseases ,Single-stranded oligonucleotides ,MSH3 ,MSH2 ,Gene Targeting ,Microsatellite instability ,DNA mismatch repair ,Targeted single-base substitution ,MutL Protein Homolog 1 - Abstract
a b s t r a c t 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.
- Published
- 2011