Tetracycline-inducible gene expression in Trichomonas vaginalis.
Transient and stable gene delivery systems are available for Trichomonas vaginalis, however, they do not allow regulated expression of target genes. To study essential genes or proteins that are toxic to the cells when over expressed, we have developed an inducible/repressible gene expression system in this parasite, which is driven by the tet-operator (tetO) and regulated tetracycline-responsive Tet repressor (TetR). Inducible chloramphenicol acetyl transferase (CAT) gene expression is observed using a concentration of tetracycline (Tc) as low as 0.1 microg x ml(-1). Expression increases with drug dose with a maximum level of CAT induction achieved in stable transfectants using 5 microg x ml(-1) Tc. CAT protein expression is detectable within 12 h and reaches a maximum level at 48 h, demonstrating that inducible expression is time and dose-dependent. In an inverse experiment, parasites previously cultivated with 1 microg x ml(-1) of Tc for 48 h, were grown in the absence of drug to determine the kinetics of repression. A significant decrease in protein concentration is detected after 48 h, and no detectable protein is observed after 72 h. Experiments replacing the CAT gene with the puromycin N-acetyltransferase (PAC) gene in the Tet regulated expression construct have demonstrated the use of this system for testing putative toxic and essential genes. The establishment of regulated gene expression of exogenous genes in T. vaginalis represents a crucial step towards determining the function of proteins in this divergent parasite.
Ortiz
Diana
D
Department of Microbiology, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA.
Johnson
Patricia J
PJ
D000123:Acetyltransferases / Q000235:genetics / Q000378:metabolism; D000818:Animals; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D008957:Models, Genetic; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000096:biosynthesis / Q000235:genetics; D013752:Tetracycline / Q000494:pharmacology*; D015533:Transcriptional Activation; D014246:Trichomonas vaginalis / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism
D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D000123:Acetyltransferases; C045336:puromycin N-acetyltransferase; D015500:Chloramphenicol O-Acetyltransferase; D013752:Tetracycline