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. 2000 Sep 15;28(18):e84. doi: 10.1093/nar/28.18.e84

Figure 1.

Figure 1

(A) Diagrammatical representation of the EGFP-Neo/Kan cassette used in the homologous recombination experiments. The cassette was amplified from plasmid pEGFP-N22 with primers HbbNeo at the 3′-end and either HbbEGFP, GammaEGFP or EpsilonEGFP at the 5′-end. The gene-specific 50 nt homology arms at the 5′-ends of these primers are represented by grey boxes, while the 3′-ends of these primers are targeted to amplify the EGFP-Neo/Kan cassette. Primers EGFPseq and Kanseq (blue arrows) were used to sequence across the recombination junctions of the EGFP-Neo/Kan cassette and confirm the specificity of the recombinant clones. (B) Diagram of the β-globin locus drawn to scale, illustrating the targeting of the EGFP-Neo/Kan cassette to each one of the functional globin genes. The insertion of the EGFP-Neo/Kan cassette (2.7 kb) is accompanied by deletions ranging from 1.4 to 44 kb of the intervening sequences. Correct insertion of the EGFP-Neo/Kan cassette at each one of the genes was confirmed with primers HbbR at the 3′-end and either ARMS-1, Aγ39049 or EpsilonScrnF at the 5′-end (purple arrows). Primers LUG1 and LUG2 (not shown) amplify a 440 bp fragment from the β-globin gene while primer Aγ39512 (not shown) amplifies a 464 bp product from each of the γ-globin genes with primer Aγ39049.