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Nucleic Acids Research 2005 33(3):e24; doi:10.1093/nar/gni014
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Published online 7 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

Transposon-mediated generation of targeting vectors for the production of gene knockouts

Chunfang Zhang1,*, Danny Kitsberg2, Hun Chy1, Qi Zhou2 and John R. Morrison1,3

1 CopyRat Pty Ltd 27-31 Wright Street, Clayton, Victoria 3168, Australia 2 IngenKO Pty Ltd 27-31 Wright Street, Clayton, Victoria 3168, Australia 3 Monash Institute of Reproduction and Development, Monash University 27-31 Wright Street, Clayton, Victoria 3168, Australia

*To whom correspondence should be addressed. Tel: +61 3 95947224; Fax: +61 3 95947111; Email: czhang{at}copyrat.com.au

Received October 31, 2004. Revised December 22, 2004. Accepted December 22, 2004.

Vectors used for gene targeting experiments usually consist of a selectable marker flanked by two regions of homology to the targeted gene. In a homologous recombination event, the selectable marker replaces an essential element of the target gene rendering it inactive. Other applications of gene targeting technology include gene replacement (knockins) and conditional vectors which allow for the generation of inducible or tissue-specific gene-targeting events. The assembly of gene-targeting vectors is generally a laborious process requiring considerable technical skill. The procedures presented here report the application of transposons as tools for the construction of targeting vectors. Two mini-Mu transposons were sequentially inserted by in vitro transposition at each side of the region targeted for deletion. One such transposon carries an antibiotic resistance marker suitable for selection in mammalian cells. A deletion is then generated between the two transposons either by LoxP-induced recombination or by restriction digestion followed by ligation. This deletion removes part of both transposons plus the targeted region in between, leaving a transposon carrying the selectable marker flanked by two arms which are homologous to the targeted gene. Targeting vectors constructed using these transposons were electroporated into embryonic stem cells and shown to be effective in gene-targeting events.


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M. Aoyama, K. Agari, G.-H. Sun-Wada, M. Futai, and Y. Wada
Simple and straightforward construction of a mouse gene targeting vector using in vitro transposition reactions
Nucleic Acids Res., March 22, 2005; 33(5): e52 - e52.
[Abstract] [Full Text] [PDF]


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