A New Conditional Transcription Factor for Use in Toxoplasma Gondii.

Abstract

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.