pTcINDEX: a stable tetracycline-regulated expression vector for Trypanosoma cruzi.
BACKGROUND
Trypanosoma cruzi is a protozoan pathogen of major medical importance in Latin America. It is also an early diverging eukaryote that displays many unusual biochemical features. The completion of the T. cruzi genome project has highlighted the need to extend the range of techniques available to study gene function. To this end we report the development of a stable tetracycline-dependent expression vector applicable to this parasite and describe in detail the parameters of the system.


RESULTS
We first produced T. cruzi cell lines that constitutively expressed bacteriophage T7 RNA polymerase and the tetracycline repressor protein from a multicopy episome. An integrative vector with an inducible expression site under the control of a tetracycline-regulatable T7 promoter (pTcINDEX) was targeted to the transcriptionally silent ribosomal RNA spacer region of these parasites and transformants selected using a T7 RNA polymerase-dependent hygromycin resistance gene. To test the system we used two marker proteins, luciferase and red fluorescent protein (RFP), and an endogenous parasite protein (a mitochondrial superoxide dismutase). In each case we found that induction was both time and dose-dependent. Luciferase mRNA could be induced by at least 100-fold, and luciferase activity up to 60-fold, within 24 hours of the addition of tetracycline. When we examined RFP induction by confocal microscopy and fluorescence activated cell sorter, we observed very high levels of expression (>1000-fold increase in fluorescence intensity), although this was not synchronous throughout clonal populations. Induction of superoxide dismutase resulted in an 18-fold increase in cellular activity. The observation that a tagged version of the enzyme was correctly targeted to the mitochondrion demonstrates that our expression system may also provide a high-throughput strategy for subcellular localisation.


CONCLUSIONS
Our results show that pTcINDEX represents a valuable addition to the genetic tools available for T. cruzi. The vector system is sufficiently flexible that it should have widespread uses including inducible expression of tagged proteins, generation of conditional knockout cell lines and the application of dominant-negative approaches.
Taylor
Martin C
MC
London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK. martin.c.taylor@lshtm.ac.uk
Kelly
John M
JM
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D000939:Epitopes; D005821:Genetic Techniques; D005822:Genetic Vectors; D016678:Genome; D008156:Luciferases / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D013482:Superoxide Dismutase / Q000378:metabolism; D013752:Tetracycline / Q000494:pharmacology*; D014349:Trypanosoma cruzi / Q000235:genetics* / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics*; D000097573:Red Fluorescent Protein
D012321:DNA-Directed RNA Polymerases; D000939:Epitopes; D008156:Luciferases; D008164:Luminescent Proteins; D013482:Superoxide Dismutase; D013752:Tetracycline; D014764:Viral Proteins; D000097573:Red Fluorescent Protein; C062210:bacteriophage T7 RNA polymerase