Figure 5

Homologous recombination targeting strategy. The donor construct P{>w^hs,Pld*>} (top) lacks most of the second and ninth exons and contains two stop codons and a recognition site for I-Sce I in exon 5. The extrachromosomal targeting molecule is generated by FLP recombinase-mediated excision and I-SceI cleaving of the donor construct (middle). Alignment of the targeting DNA and the endogenous Pld locus by ends-in recombination results in two mutant copies of the Pld gene (bottom). Locations of relevant restriction sites and Southern blot hybridization probes used (#1 and #2) are indicated. The sizes of the different fragments detected by Southern blot analysis of the wild-type and mutated alleles are indicated beneath the genomic representations (dotted lines). B=BamH I; N=Not I. (Inset) Southern blot analysis of the Pld targeting event. Genomic DNA was digested with BamH I and Not I, and blotted to a membrane. The membrane was hybridized with Probe #2. Lane 1, y¹w^67c23wild-type control; lane 2, DNA from the donor P{>w^hs,Pld*>} flies; lane 3, DNA from the heterozygote line 3 candidate; lanes 4–6, DNA from the homozygote lines 3, 8 and 9 candidates, respectively. The wild-type band in the yw control is observed as a doublet because of a polymorphism or insertion in one of the parental chromosomes between Pld and bin3. Southern blot hybridization using probe #1 confirmed the structure of the mutant allele at the 5' end of the insertion site for all three lines as diagrammed (not shown). None of the Pld^nulllines retained the I-SceI restriction sequence at the site of integration in exon 5: in each case, the wild-type sequence was restored.