Genomic engineering at the <i>Hairless</i> locus.

<p>(A) The <i>Hairless (H)</i> locus is flanked by the <i>CG5466</i> and <i>Pi3K92E</i> genes. The scheme depicts the major transcripts; arrowheads point in the direction of transcription. The coding region is coloured, UTRs are shown paler (according to <a href="http://www.flybase.org/" target="_blank">http://www.flybase.org</a>, released 2014_06). (B) Restriction map of the genomic <i>H</i> wild type region including the following restriction sites: B, <i>Bam</i> HI; E, <i>Eco</i> RI; H, <i>Hin</i>d III; K, <i>Kpn</i> I; N, <i>Not</i> I; V, <i>Eco</i> RV; X, <i>Xho</i> I. For the engineering, a 3 kb <i>Bam</i> HI–<i>Kpn</i> I fragment was cloned as 5’arm and a 6.4kb <i>Eco</i> RV—<i>Xho</i> I fragment as 3’arm into pGX-attP to give pGX-H; respective restriction sites are in red. The vector contains a <i>white</i>⁺ gene for selection, an attP site for site specific integration and two loxP sites for marker removal. The respective elements are not to scale. Homologous recombination at the <i>H</i> locus results in the founder knock out line (<i>H</i>^{<i>attP w+</i>}). Excision of <i>white</i>⁺ by CreI-recombinase results in the knock out line <i>H</i>^<i>attP</i>, lacking about 5.1 kb of the <i>H</i> locus, i.e. the entire coding region. Primer pairs used for genotyping are indicated; they are not to scale. (C) Fly lines were genotyped by PCR. Left panel: Homologous recombination at the <i>H</i> locus was confirmed with 5’HR PCR: primer P1 binds upstream of the 5’arm and primer P2 within attP site to give ~3.1 kb fragment in the <i>H</i>^{<i>attP w+</i>} line, but not the starting line (pGX-H). Right panel: Excision of <i>white</i>^<i>+</i> in <i>H</i>^<i>attP</i> was confirmed with H-flox PCR: using primer pair P3/P4 flanking the deletion, a ~1.3 kb fragment is seen in <i>H</i>^<i>attP</i> but not in the starting line <i>H</i>^{<i>attP w+</i>}. (M, marker λ-DNA, <i>Eco</i> RI/<i>Hin</i>d III digested; numbers are appr. size in kb). (D) Scanning electron micrographs of thoraces from wild type (WT) (left panel) in comparison to a heterozygous mutant <i>H</i>^<i>attP</i> (right panel). <i>H</i> mutants are typified by loss of bristles; arrows point to examples of macrochaetae loss. The inset shows an enlargement of an orbital bristles; note the double socket in the mutant. Scale, 100 and 20 μm, respectively.