Trypanosoma cruzi: Exogenously regulated gene expression

Wen, L.-M., Xu, P., Benegal, G., Carvaho, M. R. C., Butler, D. R., and Buck , G. A. 2001. Typanosoma cruzi: Exogenously regulated gene expression. Expe rimental Parasitology 97, 196-204. A regulated expression vector would prov ide a strong tool for the dissection of gene function in Trypanosoma cruzi. Herein, we establish a system in which genes in T cruzi expression vectors can be exogenously regulated by tetracycline. We first generated strains o f T. cruzi that stably express the repressor of the bacterial tetracycline resistance gene and T7 RNA polymerase. Based on these strains, we developed two T. cruzi expression systems regulated by tetracycline-the first by use of a regulated rRNA promoter and the second by use of a regulated T7 promo ter. In the former, we constructed an expression vector in which tetracycli ne resistance gene operators flank the transcription start point of the T. cruzi rRNA gene promoter. Reporter gene activity from this modified promote r was regulated up to 20-fold in the presence of different concentrations o f tetracycline. In the T7 system, tetracycline resistance gene operators fl ank the transcription start point of the T7 promoter. Reporter gene activit y from this modified promoter was regulated up to 150-fold in the presence of different concentrations of tetracycline. Expression in these systems wa s repressed when tetracycline was removed even after full induction for ext ended periods in the presence of tetracycline. We are now using these two s ystems to test protein function in T. cruzi. (C) 2001 Academic Press.