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. 2014 Mar 19;289(18):12578–12592. doi: 10.1074/jbc.M113.544718

FIGURE 4.

FIGURE 4.

Generation of ROP18 WT and MUT expressing type I T. gondii in rop18 knock-out background. A, the ROP18 coding sequence of a virulent type I (RH Δku80Δhxgprt) strain was replaced with TUB-promoter-ROP18-Ty1-tagged-HXGPRT by homologous recombination. B, primers were used to evaluate the generation of ROP18-WT or MUT Ty1-tagged RH strain in rop18 knock-out background. The 5′-integration was verified with primer F1 and R1; the 3′-integration was verified with primer F2 and R2; the insertion of TUB promoter-ROP18 -Ty1-HXGPRT cassette into ROP18 locus was verified with primer F1 and R2. C, expression of wild-type ROP18 and its mutant in ROP18-WT or MUT RH strains were determined by RT-PCR. Expression levels of SAG1 were analyzed and shown as loading controls. Data was representative of two independent experiments. D, expression of wild-type ROP18 and its mutant ROP18-WT or MUT RH strains were determined by Western blotting. Expression levels of SAG1 antigen were analyzed and shown as loading controls. Data are representative of two independent experiments. IB, immunoblot.