The use of Tn5 transposable elements in a gene trapping strategy for the protozoan Leishmania Eliane C. Laurentino a,1 , Jeronimo C. Ruiz a,1,2 , Loislene O. Brito a , Michael Fiandt b , Liliana M. Nicoletti a , M.C. Jamur a , C. Oliver a , Luiz R.O. Tosi a , Angela K. Cruz a, * a Departamento de Biologia Celular e Molecular e Bioagentes Patoge ˆnicos, Faculdade de Medicina de Ribeira ˜o Preto, Universidade de Sa ˜o Paulo, Av. Bandeirantes, 3900, Ribeira ˜o Preto, 14049-900 Sa ˜o Paulo, Brazil b EPICENTRE Biotechnologies, 726 Post Road, Madison, WI 53713, USA Received 25 September 2006; received in revised form 17 December 2006; accepted 22 December 2006 Abstract The use of transposable elements as a gene-trapping strategy is a powerful tool for gene discovery. Herein we describe the develop- ment of a transposable system, based on the bacterial Tn5 transposon, which has been used successfully in Leishmania braziliensis. The transposon carries the neomycin phosphotransferase gene, which is expressed only when inserted in-frame with a Leishmania gene pres- ent in the target DNA. Four cosmid clones from a L. braziliensis genomic library were used as targets in transposition reactions and four insertional libraries were constructed and transfected in L. braziliensis. Clones resistant to G418 were selected and analysed by immu- nofluorescence in order to identify the subcellular localisation of the protein coded by the trapped gene. A definitive subcellular local- isation for neomycin phosphotransferase/targeted protein fusion was not obtained in any of the four Leishmania clones investigated. However, the constructed transposable element is highly efficient considering the frequency of insertion in large targets and is therefore a useful tool for functional genetic studies in Leishmania. Our data confirm the utility of the Tn5 transposon system for insertion of sequencing priming sites into target DNA. Furthermore, the high frequency of insertion and even distribution are important in studying genomic regions bearing long and polymorphic repetitive sequences. Ó 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Leishmania braziliensis; Tn5; Transposon; In vitro transposition; Gene trapping 1. Introduction The Leishmania genus consists of more than 20 differ- ent species that are the causative agents of leishmaniasis, a human disease with a broad spectrum of clinical man- ifestation such as visceral, cutaneous or mucosal lesions. According to the World Health Organization (WHO), the different species of Leishmania parasites infect between 1.5 and 2 million people per year in tropical and temperate regions of the world (WHO, May 2000). No effective vaccines against leishmaniasis are available yet (Handman, 1997) and treatment relies on highly toxic chemotherapeutic agents (Papadopoulou and Ouellette, 1993). Over the last few years several genetic tools have been introduced which allow manipulation and analysis of the Leishmania genome (Clayton, 1999). These tools include different expression vectors, positive and negative selectable markers, reporter genes, methods for gene replacement and functional complementation, and transposon-based muta- genesis, among others (Cruz et al., 1991; LeBowitz et al., 1992, 1993; Ryan et al., 1993; Ha et al., 1996; Gueiros-Filho and Beverley, 1997; Roy et al., 2000; Tosi and Beverley, 2000; Augusto et al., 2004; Denise et al., 2004). 0020-7519/$30.00 Ó 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2006.12.021 * Corresponding author. Tel.: +55 16 3602 3318; fax: +55 16 3633 1786. E-mail address: akcruz@fmrp.usp.br (A.K. Cruz). 1 These authors contributed equally to this work. 2 Present address: Centro de Pesquisas Rene ´ Rachou, FIOCRUZ, Belo Horizonte, Brazil. www.elsevier.com/locate/ijpara International Journal for Parasitology 37 (2007) 735–742