Fig. 3

Homologous recombination in A. deanei. Genomic DNA was restricted with specified enzymes and analyzed by Southern blot hybridizations using probes against γ-amastin, δ-amastin, hyg, or neo, as indicated above each blot. The wild type (WT) and three clones (1, 2, and 3) were analyzed for Δ-γ-ama^Hyg SKO (a), Δ-γ-ama^Hyg/Δ-γ-ama^Neo DKO (b), and Δ-γ-ama^Hyg/Δ-δ-ama^Neo PKO cell lines (c). (a) In the Δ-γ-ama^Hyg SKO cell lines, a band shift of 2 kb between the γ-amastin WT locus and the recombinant locus containing hyg could be readily observed in all three clones analyzed, indicating replacement of the first γ-amastin allele by hyg; (b) a second transfection with a cassette encoding neo targeting the γ-amastin locus completely abolished the γ-amastin WT locus in all three Δ-γ-ama^Hyg/Δ-γ-ama^Neo DKO clones analyzed; as a control, gDNA from the SKO lines Δ-γ-ama^Hyg (γH) and Δ-γ-ama^Neo (γN) was tested; (c) transfection of Δ-γ-ama^Hyg SKO clone 1 with a cassette encoding neo targeting the δ-amastin locus yielded the Δ-γ-ama^Hyg/Δ-δ-ama^Neo PKO cell lines. (d) Restriction maps are provided for the various blots. The striped rectangles represent genomic regions up and downstream of the insertion sites for the cassettes, light blue and violet rectangles represent the 5′- and 3′-flanking regions (FR) of the γ- and δ-amastin genes, respectively, that are present in the cassettes; blue and orange rectangles represent the γ- and δ-amastin ORFs, respectively; and hyg and neo are represented by yellow and pink rectangles, respectively. The red bar above the inserted marker gene highlights the binding region for the DNA probe used for hybridization