Figure 3.

TgPEPCK_mt promotes the lytic cycle of glycolysis-competent tachyzoites. A, scheme depicting double homologous recombination-mediated knock-out of the TgPEPCK_mt gene by DHFR-TS in tachyzoites. The construct was transfected into the indicated parental strains, and pyrimethamine-resistant clonal parasites were screened for 5′- and 3′-crossover events using applicable primer pairs (TgPEPCK_mt-KO-5′Scr-F1/R1 or TgPEPCK_mt-KO-3′Scr-F1/R1). B, genomic PCR screening showing 5′ and 3′ recombination in selected parasite clones generated according to the scheme described in A. The specificity of the TgPEPCK_mt knock-out in recombination-positive clones was confirmed by sequencing of PCR amplicons. C, validation of the Δtgpepck_mt mutant by transcript analysis. A representative clonal mutant was examined for the presence or absence of TgPEPCK_mt and TgFBP2 (control) transcripts using ORF-specific primer sets. Parental strain was included as a positive control. D, plaque assays revealing comparative growth of the Δtgpepck_mt mutant with respect to its parental strain. Shown are the area (arbitrary units) and number of plaques (mean ± S.E. (error bars), n = 3 assays). Significance was tested using Student's t test (***, p < 0.001).