Mpa xa

Following natural egress, freshly harvested parasites were transfected with plasmids, using protocols previously described (Shen et al., 2014 (link)). In brief, ~2 × 10⁷ extracellular parasites were resuspended in 370 μL cytomix buffer were mixed with ~30 μL purified plasmid or amplicon DNA in a 4-mm gap BTX cuvette and electroporated using a BTX ECM 830 electroporator (Harvard Apparatus) using the following parameters: 1,700 V, 176-μs pulse length, 2 pulses, 100-ms interval between pulses. Transgenic parasites were isolated by outgrowth under selection with mycophenolic acid (25 μg/mL) and xanthine (50 μg/mL) (MPA/Xa), pyrimethamine (Pyr) (3 mM), chloramphenicol (20 mM), 5-fluorodeoxyuracil (10 μM) (Sigma), as needed. Stable clones were isolated by limiting dilution on HFF monolayers grown in 96-well plates.

T. gondii tachyzoites were serially passaged in human foreskin fibroblast (HFF) monolayers. Strains used in this study are listed in Table S2. Gene disruptants and complemented lines were generated using CRISPR/Cas9 (Shen et al., 2014 (link)), as described in the Supplemental materials. Stable transgenic parasites were selected in mycophenolic acid (25 μg/ml) and xanthine (50 μg/ml) (MPA/Xa) or pyrimethamine (Pyr) (3 mM) (Sigma). Cultures were negative for Mycoplasma contamination using the e-Myco plus mycoplasma PCR detection kit (Boca Scientific). U3A and U3A-STAT1 cells (Zhang et al., 2005 (link)), HeLa cells (ATCC CCL-2), 3T3 cells (ATCC CCL-163) and RAW 264.7 mouse macrophages (ATCC TIB-71) were maintained in complete media without gentamicin at 37°C in 5% CO₂.