The use of Tn5 transposable elements in a gene trapping strategy for the protozoan Leishmania.
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.
Laurentino
Eliane C
EC
Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Av Bandeirantes, Ribeirão Preto, São Paulo, Brazil.
Ruiz
Jeronimo C
JC
Brito
Loislene O
LO
Fiandt
Michael
M
Nicoletti
Liliana M
LM
Jamur
M C
MC
Oliver
C
C
Tosi
Luiz R O
LR
Cruz
Angela K
AK
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004251:DNA Transposable Elements / Q000235:genetics*; D015723:Gene Library; D023281:Genomics / Q000379:methods; D007892:Leishmania braziliensis / Q000235:genetics*; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D014162:Transfection
D004251:DNA Transposable Elements