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Nucleic Acids Research 2005 33(4):e44; doi:10.1093/nar/gni045
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Published online 1 March 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

Tagging genes with cassette-exchange sites

Gilda Cobellis1, Giancarlo Nicolaus2, Mariangela Iovino2, Antonio Romito1, Emanuele Marra2, Manlio Barbarisi1, Marco Sardiello1, Francesco P. Di Giorgio2, Nicola Iovino2, Massimo Zollo1, Andrea Ballabio1,3,* and Riccardo Cortese2

1Telethon Institute of Genetics and Medicine, Via P. Castellino 111, 80131 Naples, Italy 2Istituto di Ricerche di Biologia Molecolare P. Angeletti Via Pontina km 30,600, 00040 Pomezia, Rome, Italy 3Medical Genetics, Department of Pediatrics, Federico II University Via S. Pansini, 5, 80131 Naples, Italy

*To whom correspondence should be addressed. Tel: +39 081 6132207; Fax: +39 081 5790919; Email: ballabio{at}tigem.it

Received December 28, 2004. Revised February 15, 2005. Accepted February 15, 2005.

In an effort to make transgenesis more flexible and reproducible, we developed a system based on novel 5' and 3' ‘gene trap’ vectors containing heterospecific Flp recognition target sites and the corresponding ‘exchange’ vectors allowing the insertion of any DNA sequence of interest into the trapped locus. Flp-recombinase-mediated cassette exchange was demonstrated to be highly efficient in our system, even in the absence of locus-specific selection. The feasibility of constructing a library of ES cell clones using our gene trap vectors was tested and a thousand insertion sites were characterized, following electroporation in ES cells, by RACE–PCR and sequencing. We validated the system in vivo for two trapped loci in transgenic mice and demonstrated that the reporter transgenes inserted into the trapped loci have an expression pattern identical to the endogenous genes. We believe that this system will facilitate in vivo studies of gene function and large-scale generation of mouse models of human diseases, caused by not only loss but also gain of function alleles.

Corresponding may also be addressed to Riccardo Cortese. Tel: +39 06 910932200; Fax: +39 06 91093200; Email: riccardo_cortese{at}merck.com

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors


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