Vector constructs.

All vectors were generated using the MLV-based vector LNSX indicated at the top,34 which expresses Neo from the promoter in the 5′ long-terminal repeat (LTR). Coding elements from the human^Aγ-globin gene (exons, filled boxes; introns and 3′ untranslated regions, filled bars; polyadenylation site, pA) were inserted in the opposite orientation with respect to virus transcription, contain a 714-bp internal deletion of intron 2, and are transcribed from a −127-bp β-globin promoter (striped box) as previously described.14,15 Vector HS40-5 contains the α-globin HS-40 enhancer (stippled box) in the double-copy position of the 3′ LTR, from which it is copied into the 5′ LTR during provirus integration. All other vectors incorporate the HS-40 enhancer immediately adjacent to the β-globin promoter. The γ-globin cassette extends 277 bp 3′ of exon 3 in vectors HS40-5 and -9, and 470 bp 3′ of exon 3 in vectors HS40-6, -10, and -11. Vectors HS40-10 and HS40-11 also contain a 58-bp deletion within the extended 3′ region. Vector HS40-11 has a 1.2-kb fragment (graded box) containing the cHS4 chromatin insulator integrated in the double-copy position of the 3′LTR.27 Critical restriction sites: B, BamHI; A, AvrII; S, StuI; N, NheI; R,RsaI; H, HindIII.