FIG 5.

Insertion of DHFR* and mCherry cassettes into the orthologue of TGVAND_290860 using SNF selection. (A and B) Diagram of DHFR* (A) and mCherry (B) integration into the TGVAND_290860 orthologue. PCR primers used are indicated with arrows in the diagram and above the top of the gel. Shown is an agarose gel separation of PCR products detecting integration in an SNF^r clone or RH (negative control). GRA1p, Gra1 promoter/positive control for PCR. Asterisks mark lanes with unspecific bands. (C) Survival plot showing that disruption of TGVAND_290860 in the mCherry-positive SNF^r clone does not affect virulence in CD-1 mice. Mice (n = 5) were inoculated with 1,000 tachyzoites i.p. The inset shows fluorescence of mCherry-positive parasites. Red, mCherry; blue, Hoechst-stained nuclei. (D) Representative plaques from a plaque assay of wild-type RH and an mCherry-positive SNF^r clone.