A New Conditional Transcription Factor for Use in <i>Toxoplasma Gondii</i>

Toxoplasma gondii is an intracellular eukaryotic parasite in the phylum Apicomplexa. Reversible, conditional control of gene transcription was achieved previously in this organism using a tet-off system that was controlled by tetracycline. Although revolutionary for the field at the time, that system in Toxoplasma yielded modest signal-to-noise ratios (SNR) and proved to be leaky in the "off" state (i.e., it suffered from high background expression). Here we report the development of a new reversible and robust Conditional Transcription Factor (CTF) that is controlled by rapamycin. Although we originally intended to design a dimerizable drug-on transcription control system, the CTF unexpectedly functions in a drug-off fashion. In stably-transfected tachyzoites, CTF regulates a fluorescent reporter gene in Toxoplasma to achieve almost no background expression in the presence of rapamycin (i.e. near-zero leakiness), while in the absence of rapamycin 84% of tachyzoites express EYFP in comparison with 87% of constitutively-expressed positive controls, with 59% mean fluorescence intensity relative to positive controls. Conditional regulation of the median fluorescence intensity of the reporter gene reached an impressive SNR averaging 1489, approximating an "on/off" switch. The modular design of the CTF is expected to facilitate application to varied genes, incremental modifications, and adaptation for use in other apicomplexan organisms.