Chemical Mutagenesis of Toxoplasma Parasites

Parasites were chemically mutagenized as previously described, with the following modifications [37 (link)]. Briefly, ~10⁷ tachyzoites (RH strain) growing intracellularly in HFF cells in a T25 flask were incubated at 37°C for 4 h in 0.1% Fetal Bovine Serum (FBS) DMEM growth medium containing either 2.5 mM ethyl methane sulphonate (EMS) or the appropriate vehicle controls. After exposure to the mutagen, parasites were washed three times with 1XPBS. The mutagenized population was allowed to recover in a fresh T25 flask containing an HFF monolayer without the drug for 3–5 days. Released tachyzoites were then inoculated into fresh cell monolayers in a medium containing 100 nM L35 and incubated until viable extracellular tachyzoites emerged 8–10 days later. Surviving parasites were passaged once more under continued L35 treatment and cloned by limiting dilution. Four cloned mutants were isolated, each from 6 independent mutagenesis experiments. Thus, each flask contained unique SNV pools.