Figure 4.

Generation of CN gene knock-out mutants. A, Schematic diagram of genomic loci for wild-type and knock-out alleles of CanA-14F, Pp2B–14D, and CanA1. CanA-14F^KO and Pp2B–14D^KO constructs precisely remove all of the protein coding regions of the corresponding genes, while the CanA1^KO construct removes the second and third exons. B, Verification of knock-outs. Knock-out lines were confirmed by PCR, shown schematically in the upper panel. Two separate PCRs were performed per knock-out candidate, one corresponding to the 5′ end of the knock-out locus and the second corresponding to the 3′ end. Internal primers were made bridging the loxP and white regions, while external primers were made to areas outside the homologous region. Knock-out lines show bands of the expected size (KO), while P{donor} lines, where the knock-out cassette is integrated at a non-homologous site, show no bands (Donor). CanB^KO and CanB2^KO were made using the same strategy, and will be described elsewhere.