Figure 1. Generation of a TgPhIL1 knockout parasite line.

(A) A knockout construct containing a ble cassette, flanked by 3 kb of upstream and downstream noncoding sequence from the TgPhIL1 genomic locus was linearized and transfected into wild-type RH parasites, where it was predicted to integrate into the TgPhIL1 locus by homologous recombination. (B) After three rounds of selection with phleomycin, immunofluorescence with anti-TgPhIL1 antiserum (red) revealed that 6% of the parasites were negative for TgPhIL1, indicating successful disruption of the open reading frame. A combined phase contrast and immunofluorescence image is shown. Scale bar  = 5 µm. (C) Western blotting with anti-TgPhIL1 confirmed the absence of TgPhIL1 expression in the knockout parasites (RHΔTgPhIL1) and restoration of TgPhIL1 expression to approximately wild-type (RH) levels in the complemented clones (RHΔTgPhIL1/PhIL1-C5, C6, C7). The blot was simultaneously probed with anti-actin as a loading control. As seen here, TgPhIL1 sometimes resolves by SDS-PAGE as a tightly spaced doublet, possibly reflecting the presence of an as yet uncharacterized posttranslational modification. Numbers on the left indicate molecular mass in kDa.