SpringerProtocols: Abstract: Gene Modification in Embryonic Stem Cells by Single-Stranded DNA Oligonucleotides

Gene Modification in Embryonic Stem Cells by Single-Stranded DNA Oligonucleotides

By: Marieke Aarts³, Marleen Dekker³, Rob Dekker³, Sandra de Vries³, Anja van der Wal³, Eva Wielders³, Hein te Riele³

Abstract

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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.

Affiliation(s): (3) Division of Molecular Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands

Book Title: Gene Knockout Protocols: Second Edition

Series: Methods in Molecular Biology | Volume: 530 | Pub. Date: Jun-01-2008 | Page Range: 79-99 | DOI: 10.1007/978-1-59745-471-1_5

Subject: Genetics/Genomics

Key Words: Subtle gene modification - single-stranded oligonucleotides - DNA mismatch repair - codon substitution - mouse mutant - gene therapy

References

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