Experimental Parasitology

Wen, L.-M., Xu, P., Benegal, G., Carvaho, M. R. C., Butler, D. R., and Buck, G. A. 2001. Trypanosoma cruzi: Exogenously regulated gene expression. Experimental Parasitology 97, 196–204. A regulated expression vector would provide 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 of 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 promoter. In the former, we constructed an expression vector in which tetracycline resistance gene operators flank the transcription start point of the T. cruzi rRNA gene promoter. Reporter gene activity from this modified promoter was regulated up to 20-fold in the presence of different concentrations of tetracycline. In the T7 system, tetracycline resistance gene operators flank the transcription start point of the T7 promoter. Reporter gene activity from this modified promoter was regulated up to 150-fold in the presence of different concentrations of tetracycline. Expression in these systems was repressed when tetracycline was removed even after full induction for extended periods in the presence of tetracycline. We are now using these two systems to test protein function in T. cruzi. q 2001 Academic Press Index Descriptors and Abbreviations: tetracycline-regulated gene expression; rRNA promoter; T7 promoter; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assays; rRNA, ribosomal RNA; Tet, tetracycline; TetO, tetracycline operator; TetR, tetracycline repressor; Trypanosoma cruzi; PBS, phosphate-buffered saline; PMSF, phenylmethyl sulfonyl fluoride.