Leishmania chagasi: A tetracycline-inducible cell line driven by T7 RNA polymerase

Abstract

Trypanosomatid protozoa lack consensus promoters for RNA polymerase (RNAP) II. However, the artificial insertion of the T7 promoter (P T7 ) and the tetracycline repressor into Trypanosoma brucei cell lines expressing T7RNAP allows P T7 -driven gene expression to be tetracycline-inducible. These cell lines provide a molecular tool to address protein function by several recombinant approaches. We describe here the development of an analogous Leishmania chagasi cell line bearing the genes for exogenous T7RNAP and the tetracycline repressor inserted in the multi-gene α-tubulin locus. A plasmid construct with P T7 and the tetracycline operator upstream of a reporter gene, when introduced into this cell line as episomal plasmids or chromosomal insertion into the noncoding strand of an 18SrRNA gene, resulted in tetracyclineinducible expression of the reporter as much as 16 and 150 fold, respectively. The reporter was under a much tighter control when chromosomally inserted than extra-chromosomally born. Furthermore, P T7 augmented the reporter's expression 2 fold more in comparison to P T7 -less constructs. This cell line is the first Leishmania spp. that allows the exogenous T7RNAP-driven gene expression to be tetracycline-inducible; and may provide a useful tool for addressing protein function by manipulating expression levels of Leishmania endogenous genes.

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