The ankyrin insulator improves translation of the β-globin gene, and its sequence is conserved at the site of integration.

<p>(A) Expression of the chimeric Hb (expressed as a percentage) after HMBA differentiation, in MEL cell clones carrying one VCN of T9W or AnkT9W, and T9Ank2W. One way anova test, p = 0.048. (B) The β-globin mRNA (bottom panel), expressed by T9W (VCN = 0.87,1.4), AnkT9W (VCN = 0.39, 1.07, 1.47) and T9Ank2W (VCN = 1.26, 1.98) was not detected in non-erythroid cells (B-16 melanoma) but only in differentiated MEL cells (AnkT9W, VCN = 2), indicating that the transgene's transcription is tissue specific. (C) Two amplicons were amplified by PCR corresponding to the ankyrin sequence present, respectively, in the 5′ and 3′ viral LTRs. Oligonucleotides were designed to include the ankyrin element and selectively amplify fragments either from the 5′ or 3′ LTR. Only clones bearing a single AnkT9W integrant were used. The amplified fragments exhibited the expected size of 248 bp and 231 bp, indicating that the ankyrin element was faithfully replicated in both LTRs. (D) Southern blot assay of genomic DNA from single integrant clones of MEL cells carrying either T9W or AnkT9W or from MEL control. The genomic DNAs were digested with <i>Xmn</i>I restriction enzyme, which yielded the full β-globin cassette, identified by hybridization using a β-globin <i>BamH</i>I-<i>Nco</i>I probe. Clone #6 of the AnkT9W series was reloaded in higher quantity (left panel) to amplify the signal of the transgenic human β-globin gene, because it was insufficiently loaded and exhibited a weak signal on the first blot (right panel).