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Nucleic Acids Research 2005 33(20):e174; doi:10.1093/nar/gni173
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Published online 10 November 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}oxfordjournals.org

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Towards systematic identification of Plasmodium essential genes by transposon shuttle mutagenesis

Hiroshi Sakamoto, Sabine Thiberge, Susan Akerman, Chris J. Janse1, Teresa Gil Carvalho and Robert Ménard*

Unité de Biologie et Génétique du Paludisme, Institut Pasteur 28 Rue du Dr Roux, 75724 Paris cedex 15, France 1Department of Parasitology, Leiden University Medical Centre PO Box 9600, 2300 RC Leiden, The Netherlands

*To whom correspondence should be addressed. Tel: +33 1 40 61 32 60; Fax: +33 1 40 61 30 89; Email: rmenard{at}pasteur.fr

Received September 19, 2005. Revised October 18, 2005. Accepted October 18, 2005.

After the deciphering of the genome sequences of several Plasmodium species, efforts must turn to elucidating gene function and identifying essential gene products. However, random approaches are lacking and gene targeting is inefficient in Plasmodium. Here, we established shuttle transposon mutagenesis in Plasmodium berghei. We constructed a mini-Tn5 derivative that can transpose into parasite genes cloned in Escherichia coli, providing an efficient means of generating knockout fragments. A 104-fold increase in frequencies of double-crossover homologous recombination in the parasite using a new electroporation technology permits to reproducibly generate pools of distinct mutants after transfection with mini-Tn5-interrupted sequences. The procedure opens the way to the systematic identification of essential genes in Plasmodium.


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