Fig. 1. Disruption of the Pvr locus through homologous recombination-mediated gene targeting. (A) A P-element containing 4.5 kb of Pvr genomic sequence, a mini-white gene and FRT sequences was randomly inserted by transposition onto the third chromosome. This DNA was circularized through expression of FLP recombinase and linearized through expression of the yeast restriction endonuclease I-SceI. This linear fragment recombined with the endogenous Pvr locus to produce two tandem partial copies of the Pvr gene. White boxes indicate exons that originate from the donor fragment. Black boxes indicate exons that originate from the endogenous locus. (B) PCR analysis of homologous recombinants using the primer pairs indicated in A. One primer of each pair anneals to DNA in the mini-white gene in the targeting construct, while the other primer of each pair anneals to genomic DNA from the endogenous locus. In the top panel, primers A and B were used to amplify DNA to the left of the mini-white gene. In the center panel, primers C and D were used to amplify DNA to the right of the mini-white gene. As expected, DNA from wild-type flies or flies with the donor P-element yield no PCR product in either case. DNA from homozygous Pvr knock-out embryos gives the expected size PCR products. In the bottom panel, control primers were used to amplify a fragment from the same genomic DNA used in the top and center panels. (C) Southern blot analysis of homologous recombinants. The locations and sizes of the fragments recognized by the probe are indicated in A. Red arrowhead, homologous recombinant-specific band; green arrowhead, endogenous Pvr locus band; blue arrowhead, pre-recombination donor-specific band. As Pvr^KO2, Pvr^KO4, and Pvr^KO10 are homozygous lethal early in development, DNA was obtained from adults heterozygous for each Pvr^KO allele and a wild-type copy of Pvr on a CyO chromosome. As expected, the endogenous Pvr locus band (green) is approximately twice as intense as the homologous recombinant-specific band (red), because the endogenous Pvr locus band is generated from both alleles.