FIGURE 2.

Targeted deletion of TgCPL. A, schematic illustration of the TgCPL knock-out strategy. A knock-out construct consisting of ∼3 kb of 5′- and 3′-flanking sequence from the TgCPL gene appended to either side of a DHFR-TS-selectable marker cassette was transfected into RH and Ku80 parasites for double crossover gene replacement of TgCPL. The arrows indicate PCR primers used in B. B, agarose gel electorphoresis of PCR products derived from parental (RH and Ku80) and knock-out (RHΔcpl and Ku80Δcpl) strains by amplification with the indicated primers. C, immunoblot analysis of parental and knock-out strains probed with RαTgCPL. Note the absence of the TgCPL reactivity. Asterisks denote nonspecific bands. A parallel blot was probed with anti-actin as a loading control. D, indirect immunofluorescence assay of newly invaded intracellular tachyzoites showing MαTgCPL reactivity with RH and Ku80 (arrows) but lack of reactivity with RHΔcpl or Ku80Δcpl.