A GRA2 minimal promoter improves the efficiency of TATi / Tet-Off conditional regulation of gene expression in Toxoplasma gondii.

A tetracycline-responsive transcription system (Tet-Off) adapted for use in Toxoplasma gondii (nicknamed TATi) is useful for molecular biological studies of this organism. Previous studies using TATi incorporated minimal promoters derived from the gene promoters for TgSAG1 or TgSAG4. The present study achieves improved activation and suppression of an integrated reporter gene in the absence and presence of anhydrotetracycline, respectively (p < 0.0001), by use of a newly derived minimal promoter based on the core promoter of TgGRA2. In comparison with the SAG1 minimal promoter, use of the GRA2 minimal promoter in stable transfectants has a 23-fold higher Signal to Noise Ratio for EYFP fluorescence in the absence or presence of anhydrotetracycline. We conclude that the performance of TATi for both activation and suppression of transcription can be markedly enhanced by incorporating a GRA2 minimal promoter.