Diagram and analysis of retrovirus vector. (A) The retrovirus vector HS40-6 was generated using the MLV-based vector LNSX1 indicated at the top, which expresses Neo from the promoter in the 5′ LTR. The optimal expression cassette was inserted in the opposite orientation with respect to viral transcription. This cassette consists of a single copy of the HS-40 enhancer (graded fill), the β-globin gene promoter (open thin bar) truncated to position −127, and the genomic elements of the^Aγ-globin gene (closed thin bar) starting with the first exon (filled boxes) and containing the large internal deletion (▵) of intron 2. Heavy arrows, sites of transcription initiation; sd/sa, vector splice donor/acceptor sites; Ψ, packaging signal; pA, polyadenylation sites; K, KpnI restriction sites used for Southern analysis. (B) DNA was prepared from clones of vector-transduced MEL585 cells and analyzed for intact provirus by digestion with KpnI (which cuts once in each LTR) and probing for Neo. Controls include DNA from untransduced MEL585 cells (U) and the producer clone (P) used to generate virus supernatant. The expected position of intact provirus is indicated to the left of the panel with an arrow. The limited signal for clone no. 9 was due to a loading error. (C) RNase protection analysis for ^Aγ-globin expression in the 12 MEL585 clones transduced with the retrovirus vector. The positions of the protected fragments for^Aγ-globin (170 bp, exon 2) and murine -globin (128 bp, exon 1) are indicated to the left of the panel. Two novel protected fragments in samples no. 2, 7, and 10 are indicated by asterisks. (D) The protected fragments in (C) were quantified by Phosphorimager, and expression of the transduced ^Aγ-globin cassette is reported as a percentage per copy of endogenous murine -globin. For clones no. 2, 7, and 10, the contribution from the secondary bands are indicated by the hatched portion of the bar. Clones no. 6 and 8 are marked with (▵) to indicate they contain deleted provirus.