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Nucleic Acids Research Advance Access originally published online on November 16, 2006
Nucleic Acids Research 2006 34(21):e147; doi:10.1093/nar/gkl896
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Nucleic Acids Research, 2006, Vol. 34, No. 21 e147
© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Generation of a mouse mutant by oligonucleotide-mediated gene modification in ES cells

Marieke Aarts, Marleen Dekker, Sandra de Vries, Anja van der Wal and Hein te Riele*

Division of Molecular Biology, The Netherlands Cancer Institute Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands

*To whom correspondence should be addressed. Tel: +31 20 512 20 84; Fax: +31 20 669 13 83; Email: h.t.riele{at}nki.nl

Received September 1, 2006. Revised October 6, 2006. Accepted October 10, 2006.

Oligonucleotide-mediated gene targeting is emerging as a powerful tool for the introduction of subtle gene modifications in mouse embryonic stem (ES) cells and the generation of mutant mice. However, its efficacy is strongly suppressed by DNA mismatch repair (MMR). Here we report a simple and rapid procedure for the generation of mouse mutants using transient down regulation of the central MMR protein MSH2 by RNA interference. We demonstrate that under this condition, unmodified single-stranded DNA oligonucleotides can be used to substitute single or several nucleotides. In particular, simultaneous substitution of four adjacent nucleotides was highly efficient, providing the opportunity to substitute virtually any given codon. We have used this method to create a codon substitution (N750F) in the Rb gene of mouse ES cells and show that the oligonucleotide-modified Rb allele can be transmitted through the germ line of mice.


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