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Nucleic Acids Research 2005 33(2):e20; doi:10.1093/nar/gni022
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Published online 1 February 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oupjournals.org

Methods Online

Suppression of nonsense-mediated mRNA decay permits unbiased gene trapping in mouse embryonic stem cells

Toshiaki Shigeoka, Masashi Kawaichi and Yasumasa Ishida*

Division of Gene Function in Animals, Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST) 8916-5 Takayama-cho, Ikoma-shi, Nara 630-0192, Japan

*To whom correspondence should be addressed. Tel: +81 743 72 5531; Fax: +81 743 72 5539; Email: ishiday{at}bs.naist.jp

Received December 18, 2004. Revised January 18, 2005. Accepted January 18, 2005.

An international collaborative project has been proposed to inactivate all mouse genes in embryonic stem (ES) cells using a combination of random and targeted insertional mutagenesis techniques. Random gene trapping will be the first choice in the initial phase, and gene-targeting experiments will then be carried out to individually knockout the remaining ‘difficult-to-trap’ genes. One of the most favored techniques of random insertional mutagenesis is promoter trapping, which only disrupts actively transcribed genes. Polyadenylation (poly-A) trapping, on the other hand, can capture a broader spectrum of genes including those not expressed in the target cells, but we noticed that it inevitably selects for the vector integration into the last introns of the trapped genes. Here, we present evidence that this remarkable skewing is caused by the degradation of a selectable-marker mRNA used for poly-A trapping via an mRNA-surveillance mechanism, nonsense-mediated mRNA decay (NMD). We also report the development of a novel poly-A-trap strategy, UPATrap, which suppresses NMD of the selectable-marker mRNA and permits the trapping of transcriptionally silent genes without a bias in the vector-integration site. We believe the UPATrap technology enables a simple and straightforward approach to the unbiased inactivation of all mouse genes in ES cells.


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