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Nucleic Acids Research 2005 33(5):e52; doi:10.1093/nar/gni055
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Published online 22 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

Simple and straightforward construction of a mouse gene targeting vector using in vitro transposition reactions

Minako Aoyama, Kazuko Agari, Ge-Hong Sun-Wada, Masamitsu Futai1 and Yoh Wada*

Division of Biological Science, Institute for Scientific and Industrial Research, Osaka University and CREST, Japan Science and Technology Agency Mihogaoka 8-1, Ibaraki, Osaka 567-0047, Japan 1Futai Special Laboratory, Microbial Chemistry Research Center, Microbial Chemistry Research Foundation and CREST, Japan Science and Technology Agency 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan

*To whom correspondence should be addressed. Tel: +81 6 6879 8481; Fax: +81 6 6875 5724; Email: yohwada{at}sanken.osaka-u.ac.jp

Received February 3, 2005. Revised March 7, 2005. Accepted March 7, 2005.

In a gene targeting experiment, the generation of a targeting construct often requires complex DNA manipulations. We developed a set of cassettes and plasmids useful for creating targeting vectors to modify the mammalian genome. A positive selection marker cassette (PGK/EM7p-npt), which included dual prokaryotic and eukaryotic promoters to permit consecutive selection for recombination in Escherichia coli and then in mouse embryonic stem cells, was flanked by two FRT-loxP sequences. The PGK/EM7p-npt cassette was placed between the minimum regions of a Tn7 transposable element for insertion into another DNA by means of Tn7 transposase in vitro. We also constructed a plasmid having a loxP-Zeo-loxP cassette between the modified Tn5 outer elements. These cassettes can be integrated randomly into a given genomic DNA through the in vitro transposition reaction, thus producing a collection of genomic segments flanked by loxP sites (floxed) at various positions without the use of restriction enzymes and DNA ligase. We confirmed that this system remarkably reduced the time and labor for the construction of complex gene targeting vectors.


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