Schematic representation of the EGFP-modified β-globin EBACs used in this study.

(A) An EGFP-Neo/Kan expression cassette starting with the first codon of the EGFP gene and finishing with the last codon of the Neo/Kan gene was inserted by GET Recombination between the start codon of either the β-, δ-, ^Aγ-, ^Gγ-, or ε-globin genes and the termination codon of the β-globin gene, in the 185-kb genomic insert of pEBAC/148β which contains the intact β-globin locus. This created a series of deletions ranging in size from 1.4 kb to 44 kb, while placing the EGFP gene under the regulatory elements of the corresponding gene in the context of the β-globin locus. In the ^Gγ-^Aγ-EGFP construct, the EGFP-expression cassette was inserted between the start codon of the^Gγ- and the stop codon of the ^Aγ-globin gene, with the deletion of all intervening genomic sequences. The approximate size of the resulting genomic insert in each construct is indicated. The genomic insert in each construct was maintained as a single NotI fragment in the rare multicloning site of the pEBAC vector. (B) The complete pEBAC160G cloning vector containing a modified pUC19 in the multicloning site and the EGFP reporter gene driven by the cytomegalovirus early promoter on the backbone of the vector.