Fig. 1. Establishment of a conditional knockout for MIC8. (A) Regulation of mic8Ty expression by ATc in three independent parasite strains: T7S4-1, T7S4-5 and T7S1-4. Parasites were grown with (+) or without (–) ATc for 48 hours, fixed and stained with antibodies against Ty, or against MIC6 as a control. Pictures were taken under identical exposure conditions. (B) Immunofluorescence analysis of parasite strains T7S4-1 and T7S4-5 grown for 48 hours in the absence of ATc, as shown in A. Parasites were stained with the indicated antibodies. Strong overexpression of MIC8Ty results in partial accumulation of MIC8 within the secretory pathway (arrows). Other micronemal proteins, such as MIC6, appear not to be affected by overexpression of MIC8. (C) Homologous recombination of endogenous mic8 with a knockout construct in which a CAT-expression cassette is flanked by 1.6 kb (5'FS) and 2.5 kb (3'FS) results in a knockout locus that can be identified using analytical PCR with specific oligonucleotides, as indicated by the arrows. (D) Analytical PCR analysis to verify homologous recombination of the knockout construct in the recipient strains T7S1-4 and T7S4-1. Numbers on top of each lane indicate the respective oligonucleotide combination (see C) used in each PCR reaction. Scale bars: 25 µm (A); 5 µm (B).