Knockout of <i>Tg</i>CDPK3 by double homologous recombination.

<p>Wild-type (ΔKu80) = wild-type parasites. A) Design of ΔCDPK3-TOXOY72 cosmid construct, whereby double homologous recombination replaces the 3′ half of the genomic locus of <i>Tg</i>CDPK3 with a CAT resistance cassette. Bold line shows TOXOY72 cosmid backbone. Schematic also shows hybridization site for Southern blot probe (“Pr”), recognition site for α<i>Tg</i>CDPK3 antibody (“Ab”), and restriction sites used in Southern blotting (K = <i>Kpn</i>I, M = <i>Mlu</i>I, S = <i>Sac</i>II). B) Southern blot analyses of Δ<i>Tg</i>CDPK3. <i>Kpn</i>I digest control shows identical banding pattern between wild-type and Δ<i>Tg</i>CDPK3 parasites, whereas digestion of gDNA with <i>Sac</i>II/<i>Mlu</i>I shows the expected size drop of in banding between wild-type and Δ<i>Tg</i>CDPK3 (“*”). C) Knockout of a clonal transgenic line, Δ<i>Tg</i>CDPK3, was confirmed with α<i>Tg</i>CDPK3 antibody, showing lack of any significant banding by western blot compared to wild-type parasites. α<i>Tg</i>MIC2 loading control shows equal loading of parasite lysates.