A New Conditional Transcription Factor for Use in Toxoplasma Gondii

Sharifpour, MF; Khadiv, S; Uboldi, AD; Jayawickrama, AA; Manning, S; Whittall, J; Bottema, CDK; Ellis, JT; Howe, DK; Wilson, DW; Hemmatzadeh, F; Tonkin, CJ; McAllister, MM

Author(s): Sharifpour, MF; Khadiv, S; Uboldi, AD; Jayawickrama, AA; Manning, S; Whittall, J; Bottema, CDK; Ellis, JT; Howe, DK; Wilson, DW; Hemmatzadeh, F; Tonkin, CJ; McAllister, MM;
Details: Publication Year 2026-05,Volume 21,Issue #5,Page e70240
Journal Title: Biotechnology Journal
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.
Publisher: WIley
Keywords: *Toxoplasma/genetics/metabolism/drug effects; *Transcription Factors/genetics/metabolism; Sirolimus/pharmacology; Genes, Reporter; Luminescent Proteins/genetics/metabolism; Gene Expression Regulation/drug effects; *Protozoan Proteins/genetics/metabolism; Fkbp; apicomplexa; artificial transcription factor; conditional gene expression; gene regulation; rapamycin; signal‐to‐noise ratio
Research Division(s): Infection and Global Health
PubMed ID: 42095472
Publisher's Version: https://doi.org/10.1002/biot.70240
Open Access at Publisher's Site: https://doi.org/10.1002/biot.70240
Terms of Use/Rights Notice: Refer to copyright notice on published article.

Creation Date: 2026-05-14 03:10:45

Last Modified: 2026-05-14 03:10:54