Fig. 1. Generation and verification of a targeted debcl mutation. (A) Targeting scheme for the debcl gene. The donor construct was generated by insertional cloning of a 4 kb upstream and a 2.7 kb downstream genomic sequence of the debcl gene into the targeting vector pW25. Upon chromosomal targeting, the debcl native gene is replaced with a white⁺ marker gene. (B) Verification of the debcl-targeting event by PCR, Southern blot and RT-PCR analysis. Primer pair A and B were used to amplify both the native and the knock-out locus to confirm the replacement of debcl (4.2 kb) with the white⁺ marker gene (5.2 kb). Primer pair C and D were used to verify the right arm of recombination during the screening process for potential recombinants. Primer pair E and F were used to confirm targeted recombination of the left arm of recombination (data not shown). WT refers to the yw parental strain, wild type at the debcl locus on the second chromosome; Donor refers to the debcl donor construct on the third chromosome, wild-type at the debcl native locus on the second chromosome; debcl^{6,22,23,27,59,61} represent different debcl knock-out candidates disrupted at the native locus. debcl⁶ was a potential debcl targeted deletion, but failed the PCR screen and was therefore eliminated from further characterization analysis. An additional debcl disruption allele, debcl^47/48, was also confirmed (not shown). Southern blot analysis using genomic DNA from the indicated fly strains was used to confirm both the right (SBP1) and left (SBP2) arm of recombination. For SBP1 (BglII digest) and SBP2 (SacI digest), the black arrowheads indicate the expected 6.4 kb and 8.3 kb genomic fragments in the WT and donor strains for SBP1 and SBP2, respectively. The white arrowheads indicate aberrant genomic fragments of 9.6 kb and 5.8 kb, indicative of gene-targeted replacement, for SBP1 and SBP2, respectively. RNA from L3 larvae was used as a template to confirm abolishment of the debcl transcript in debcl^KO flies, and to confirm that transcript levels are unaffected in the neighboring genes fmo-2, CG30443 and geminin. rp49 was used as a control for RT-PCR. The white asterisk indicates the absence of the debcl transcript in mutant flies. (C) Photograph of extra scutellar bristles in debcl^KO flies (white arrow).