Fig. 1

Attempt to knock out FeSOD-A using the conventional method of gene replacement by homologous recombination. First, the knockout was evaluated by PCR, using genomic DNA of wild-type parasites and mutants, FeSOD-A^−/+ and FeSOD-A^−/−/+. The correct integration of the resistance markers a NEO and b HYG was evaluated by PCR by annealing a primer in a 3′UTR region adjacent to the cassette (primer P1) and by another primer annealing within each resistance marker sequence (primers P2 or P3). The P1 primer is located 758 bp upstream of the FeSOD-A coding sequence, since the 3′UTR homology arm is 518 bp long. The cassettes used for homologous recombination are coloured in black. c Amplification of the FeSOD-A coding sequence by PCR using primers P4 and P5. d Amplification of the FeSOD-A coding sequence plus its 3′UTR by PCR using primers P1 and P5. e Western blot analysis of FeSOD-A expression in wild-type parasites and mutants (FeSOD-A^−/+ and FeSOD-A^−/−/+) using the anti-TcFeSOD-A polyclonal antibody that recognises a 26 kDa polypeptide. f Western blot analysis of α-tubulin using a monoclonal antibody, performed as a normaliser for the protein loading. MW: molecular weight standard; bp, base pairs; NC: negative control; WT: wild-type; KDa, kilodalton