TY - JOUR
T1 - Tetracycline-inducible gene expression in Trichomonas vaginalis
AU - Ortiz, Diana
AU - Johnson, Patricia J.
N1 - Funding Information:
We thank members of the lab for helpful advice and discussion, Dr. Kirkwood Land for establishing conditions to use PAC as a selectable marker, Maria Delgadillo for help with plasmid construction and Dr. Sabrina Dyall and Dr. Felix Bastida for critically reading the manuscript. This work was supported by grants (AI30537 and AI27857) from the National Institutes of Health and a Burroughs-Wellcome Scholar Award in Molecular Parasitology, of which P.J.J. is a recipient.
PY - 2003/4/25
Y1 - 2003/4/25
N2 - 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 0.1μgml-1. Expression increases with drug dose with a maximum level of CAT induction achieved in stable transfectants using 5μgml-1 Tc. CAT protein expression is detectable within 12h and reaches a maximum level at 48h, demonstrating that inducible expression is time and dose-dependent. In an inverse experiment, parasites previously cultivated with 1μgml-1 of Tc for 48h, were grown in the absence of drug to determine the kinetics of repression. A significant decrease in protein concentration is detected after 48h, and no detectable protein is observed after 72h. 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.
AB - 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 0.1μgml-1. Expression increases with drug dose with a maximum level of CAT induction achieved in stable transfectants using 5μgml-1 Tc. CAT protein expression is detectable within 12h and reaches a maximum level at 48h, demonstrating that inducible expression is time and dose-dependent. In an inverse experiment, parasites previously cultivated with 1μgml-1 of Tc for 48h, were grown in the absence of drug to determine the kinetics of repression. A significant decrease in protein concentration is detected after 48h, and no detectable protein is observed after 72h. 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.
KW - Inducible gene expression
KW - Tetracycline
KW - Trichomonas vaginalis
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U2 - 10.1016/S0166-6851(03)00043-4
DO - 10.1016/S0166-6851(03)00043-4
M3 - Article
C2 - 12706795
AN - SCOPUS:0037466355
SN - 0166-6851
VL - 128
SP - 43
EP - 49
JO - Molecular and Biochemical Parasitology
JF - Molecular and Biochemical Parasitology
IS - 1
ER -