Genetic Modification and Virulence Testing of T. gondii

The RH Δhxgprt (RH), TATi [66 (link)], DiCre-T2A [47 (link)] and RH Δku80 strains were used to construct genetically modified strains. All parasite strains were maintained in human foreskin fibroblast (HFF) cells (purchased from ATCC, USA), which were cultured in DMEM medium (Life Technologies, USA) containing 2% fetal bovine serum and 1% penicillin−streptomycin (Life Technologies, USA). Anhydrotetracycline (ATc) (Takara Bio USA, Inc., USA) at final concentration of 1 μg/mL was used to suppress the expression of pS1O7 regulated genes. Final concentration of 50 nM rapamycin (Aladdin, China) was used to induce excision of target genes engineered in the DiCre-T2A system. 10 mM mevalonate was used to induce IPP/DMAPP synthesis via the MVA pathway in the iTPI2compMVA strain. Seven-week-old female ICR mice were used for virulence tests of T. gondii strains. Nine-week-old Kunming mice were used to produce polyclonal antibodies against PGK2.