Fig. 8. (A) Generation of TTA1-knockout procyclics. (Top) A restriction map of TTA1 and its flanking regions. Three probes (U, I, and D) for Southern blotting are also indicated. (Middle) Two targeting constructs in which the entire TTA1 gene is replaced with neomycin (NEO)- or blasticidin (BSD)-resistance gene. (Bottom) Expected disrupted alleles. Predicted sizes of fragments digested with SphI, EcoRI, and SpeI, which hybridized with probes U, I, and D, respectevely, are indicated above each allele. Note that probe I should hybridize only with the wild allele. (B) Southern blot analysis of TTA1-knockout procyclics. (Left, Center, and Right) Blots with upstream (probe U), intragene (probe I), and downstream (probe D) probes, respectively. Lanes 1, wild type; lanes 2, blasticidin-resistant clone; lanes 3, geneticin-resistant clone; lanes 4, TTA1-knockout clone. Identities of fragments and size markers are shown.