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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]
Número total de Autores: 9
Tipo de documento: Artigo Científico
Fonte: International Journal for Parasitology; v. 37, n. 7, p. 735-742, June 2007.
Área do conhecimento: Ciências Biológicas - Bioquímica
Assunto(s):Protozoa   Leishmania braziliensis   Genomas   Elementos de DNA transponíveis

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)

Processo FAPESP: 00/10059-2 - Introdução a genômica comparativa e funcional de Leishmania braziliensis através do uso de transposons
Beneficiário:Eliane Cristina Laurentino
Modalidade de apoio: Bolsas no Brasil - Doutorado
Processo FAPESP: 99/12403-3 - Do genoma à biologia de Leishmania: uma abordagem compreensiva
Beneficiário:Angela Kaysel Cruz
Modalidade de apoio: Auxílio à Pesquisa - Temático
Processo FAPESP: 01/13461-9 - Abordagens de genomica comparativa atraves da utilizacao de gsss e sequenciamento do cromossomo 2 de leishmania major.
Beneficiário:Jeronimo Conceição Ruiz
Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado