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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

The use of Tn5 transposable elements in a gene trapping strategy for the protozoan Leishmania

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Laurentino, Eliane C. ; Ruiz, Jeronimo C. ; Brito, Loislene O. ; Fiandt, Michael ; Nicoletti, Liliana M. ; Jamur, M. C. ; Oliver, C. ; Tosi, Luiz R. O. ; Cruz, Angela K. [9]
Total Authors: 9
Document type: Journal article
Source: International Journal for Parasitology; v. 37, n. 7, p. 735-742, June 2007.
Field of knowledge: Biological Sciences - Biochemistry

The use of transposable elements as a gene-trapping strategy is a powerful tool for gene discovery. Herein we describe the development 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 present 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 immunofluorescence in order to identify the subcellular localisation of the protein coded by the trapped gene. A definitive subcellular localisation 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. (AU)

FAPESP's process: 99/12403-3 - From the genome to the biology of Leishmania: a comprehensive approach
Grantee:Angela Kaysel Cruz
Support Opportunities: Research Projects - Thematic Grants