Fig 3.

Truncation of TgFRM3 by a knock-in strategy. (A) Insertion of three Ty epitope tags upstream from the FH2 domain of TgFRM3 by single homologous recombination (knock-in). The schematic representation shows the TgFRM3 locus, the gene-targeting construct for gene disruption by single homologous recombination, and the locus resulting from integration of the knock-in construct. The HXGPRT resistance cassette is in blue, the TgFRM3 gene in green, the genomic region coding for TgFRM3 C terminus is in hatched green, the FH2 domain in violet, and the 3 Ty tags in red. The primers used for PCR analysis are indicated. (B) PCR analysis performed on pool-HXGPRT⁺, showing that single homologous recombination occurred. Genomic DNA from ku80-ko parasites was used as negative control. (C) Immunofluorescence assays performed on transgenic parasites using the monoclonal anti-Ty antibodies. GAP45 was used as a peripheral marker. Scale bars represent 2 μm. (D) Western blot analysis performed on transgenic or ku80-ko parasite lysates probed with anti-Ty antibodies. A doublet at around 170 kDa was detected, corresponding to the truncated NtFRM3Ty.