Generation of Complemented T. gondii Atg8 Cell Lines

Tachyzoites of the T. gondii cKD TgAtg8 cell line (12 (link)), as well as derived transgenic parasites generated in this study, were maintained by serial passage in a human foreskin fibroblast (HFF; American Type Culture Collection; CRL 1634) cell monolayer grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco), supplemented with 5% decomplemented fetal bovine serum, 2-mM l-glutamine, and a cocktail of penicillin-streptomycin at 100 μg/mL.
Complemented cell lines were generated by insertion of an additional TgAtg8 copy at the uracil phosphoribosyltransferase (UPRT) locus in the cKD TgAtg8 mutant (12 (link)). The pGFP-TgAtg8 plasmid (64 (link)) was used as a template with primers ML2463 and ML2464, and the products were self-ligated to generate p-GFP-TgAtg8Δ68-76 excluding amino acids 68 to 76 (QCAQNSGLP). A 1.5-kbp sequence corresponding to the promoter region of TgAtg8 was obtained by PCR from genomic DNA with primers ML2429 and ML2430 and cloned with NsiI upstream of the GFP-TgAtg8 or GFP-TgAtg8ΔLoop fragment in its respective plasmid. These were then used as a PCR template to amplify, with primers ML2624 and ML2625, a cassette containing the TgAtg8 promoter followed by the sequence coding for GFP-fused wild-type (WT) or truncated TgAtg8. These cassettes were cloned using NotI and XmaI into the pUPRT-TUB-Ty plasmid (65 (link)) to yield the pUPRT-GFP-TgAtg8 and pUPRT-GFP-TgAtg8ΔLoop plasmids, respectively. These plasmids were then linearized with KpnI and BamHI prior to transfection into the cKD TgAtg8 cell line (12 (link)) together with a plasmid expressing Cas9 and a UPRT-specific guide RNA under the control of a U6 promoter (66 (link)). Then transgenic parasites were selected with 5 μM fluorodeoxyuridine and cloned by limiting dilution. Primers are listed in Table S1.