Gene pulser 2 instrument

5 × 10⁶ Raji cells were suspended in 250 μl Opti-MEM I medium (Thermo Fisher Scientific), 5–10 μg plasmid DNA was added, and the cells were incubated on ice for 15 min. Electroporation (gene pulser II instrument; Bio-Rad) was performed in 4-mm cuvettes at 230 V and 975 μF. The cells were resuspended with 400 μl FCS, transferred to 5 ml fully supplemented medium as described above, and cultivated at 37°C for 2 d. For the establishment of single-cell clones, the cells were diluted in 96-well cluster plates and cultivated under selection for 4 wk. The medium was changed when necessary, and outgrowing cells were expanded. GFP expression was monitored by flow cytometry with a FACS Canto instrument by Becton Dickinson.

The human resistin promoter segment was PCR-amplified using genomic templet DNA isolated from genotyped donors. The fragments were then cloned in into the PGL3 basic plasmid using Mlu I and Bgl II restriction sites (Supplementary File 3). For transfection, U937 cells were maintained in RPMI 1640 media with 10% fetal calf serum and antibiotics. Exponentially growing cells were harvested and resuspended at a density of 2 × 10⁷/ml in RPMI media without FCS supplemented with 10 mM Dextrose and 0.1 mM DTT. 0.3 ml of the cell suspension was used for per electroporation in 0.4 cm cuvettes. For electroporation, 10 ug of reporter Plasmid and 1 ug of renilla luciferase (10:1) were mixed with 0.3 ml of cell suspension and electroporated using Bio-Rad Gene pulser II instrument (220 V and 960 uF capacitance). 48 h post electroporation, cells were lysed in 1x PLB and analyzed for luciferase and renilla activity using the Dual-Glow luciferase assay kit GloMax® Luminomete (Promega Corporation).

To complement the GRA15-KO parasites, the GRA15 coding region and putative promoter region (1,940 bp upstream of the start codon) were amplified from Pru T. gondii genomic DNA using primers TgGRA15_F and TgGRA15_R (Table S1), subcloned into pCR-Blunt II TOPO (Thermo Fisher), and sequenced. The plasmid was cut by BamHI and PacI, and the fragment containing the gra15 promoter and GRA15 coding region was cloned into the BamHI and PacI sites of the pS1K_DsRed vector, which express DsRed-Express (Clontech) under the control of the T. gondii SAG1 promoter (60 (link)). GRA15-KO Pru T. gondii parasites were filtered, washed, and resuspended in Cytomix (10 mM KPO₄, 120 mM KCl, 0.15 mM CaCl₂, 5 mM MgCl₂, 25 mM HEPES, 2 mM EDTA). Parasites were mixed with 50 µg of pS1K_DsRed/empty vector or pSIK_DsRed/gra15-promoter GRA15 expression vector supplemented with 2 mM ATP and 5 mM GSH. Parasites were electroporated by the use of a Gene Pulser II instrument (Bio-Rad Laboratories). After 48 h posttransfection, red fluorescent protein (RFP)-positive parasites were sorted using a FACS AriaIII cell sorter (BD). And then parasites were plated in limiting dilution in 96-well plates to isolate a single clone. To confirm the expression of the gene encoding GRA15, we analyzed mRNA of GRA15 from GRA15-KO parasites with empty or GRA15 expression vectors by quantitative RT-PCR.