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Nucleic Acids Research Advance Access published online on August 19, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp640
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© The Author 2009. Published by Oxford University Press.
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

Expression-independent gene trap vectors for random and targeted mutagenesis in embryonic stem cells

Anestis Tsakiridis1,2, Elena Tzouanacou1, Afifah Rahman1, Douglas Colby1, Richard Axton2, Ian Chambers1, Valerie Wilson1, Lesley Forrester2 and Joshua M. Brickman1,*

1MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, King's Buildings, West Mains Road and 2MRC Centre for Regenerative Medicine, Queens Medical Research Institute, University of Edinburgh, 47 Little France Crescent, Edinburgh, UK

*To whom correspondence should be addressed. Tel: +44 131 650 5866; Fax: +44 131 650 7773; Email: josh.brickman{at}ed.ac.uk

Received April 10, 2009. Revised July 13, 2009. Accepted July 17, 2009.

Promoterless gene trap vectors have been widely used for high-efficiency gene targeting and random mutagenesis in embryonic stem (ES) cells. Unfortunately, such vectors are only effective for genes expressed in ES cells and this has prompted the development of expression-independent vectors. These polyadenylation (poly A) trap vectors employ a splice donor to capture an endogenous gene's polyadenylation sequence and provide transcript stability. However, the spectrum of mutations generated by these vectors appears largely restricted to the last intron of target loci due to nonsense-mediated mRNA decay (NMD) making them unsuitable for gene targeting applications. Here, we present novel poly A trap vectors that overcome the effect of NMD and also employ RNA instability sequences to improve splicing efficiency. The set of random insertions generated with these vectors show a significantly reduced insertional bias and the vectors can be targeted directly to a 5' intron. We also show that this relative positional independence is linked to the human β-actin promoter and is most likely a result of its transcriptional activity in ES cells. Taken together our data indicate that these vectors are an effective tool for insertional mutagenesis that can be used for either gene trapping or gene targeting.


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