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.