JBC Invitrogen Ultrasensitive Cytokine Assays

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Originally published In Press as doi:10.1074/jbc.C100766200 on January 22, 2002

J. Biol. Chem., Vol. 277, Issue 11, 8759-8762, March 15, 2002
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ACCELERATED PUBLICATION
piggyBac-mediated Germline Transformation of the Malaria Mosquito Anopheles stephensi Using the Red Fluorescent Protein dsRED as a Selectable Marker*

Tony NolanDagger , Tom M. Bower§, Anthony E. Brown, Andrea Crisanti, and Flaminia Catteruccia§

From the Department of Biological Sciences, Sir Alexander Fleming Building, Imperial College of Science, Technology and Medicine, Imperial College Road, London SW7 2AZ, United Kingdom

It is estimated that every year malaria infects ~300 million people and accounts for the death of 2 million individuals. The Plasmodium parasites that cause malaria in humans are transmitted exclusively by mosquito species belonging to the Anopheles genus. The recent development of a gene transfer technology for Anopheles stephensi mosquitoes, using the Minos transposable element marked with the enhanced green fluorescent protein EGFP (Catteruccia, F., Nolan, T., Loukeris, T. G., Blass, C., Savakis, C., Kafatos, F. C., and Crisanti, A. (2000) Nature 405, 959-962), provides now a powerful tool to investigate the role of mosquito molecules involved in the interaction with the malaria parasite. Such technology, when further developed with additional markers and transposable elements, will be invaluable for analyzing the biology of the vector and for developing malaria-resistant mosquitoes to be used as a tool to control malaria transmission in the field. We report here the germline transformation of A. stephensi mosquitoes using a piggyBac-based transposon to drive integration of the gene encoding for the red fluorescent protein dsRED. A. stephensi embryos were injected with transformation vector pPBRED containing the dsRED marker cloned within the arms of piggyBac. Microscopic analysis of G1 larvae revealed the presence of seven fluorescent phenotypes whose different molecular origins were confirmed by Southern blotting analysis. Sequencing of the insertion sites in two lines demonstrated that integrations had occurred at TTAA nucleotides in accordance with piggyBac-mediated transpositions.

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported by a network grant of the Training and Mobility Program of the European Community.

§ Supported by the Wellcome Trust.

To whom correspondence should be addressed. Tel.: 44-20-75945412; Fax: 44-20-75945439; E-mail: f.catteruccia@ic.ac.uk.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.


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