FIG. 3.
The 1.6-kb MINE impairs enhancer-promoter interaction and functions as a barrier element in colony assays. The ability of MINE to support expression or interfere with enhancer activity was determined using reporter constructs that contain a neomycin-selectable marker gene driven by the human T-cell receptor δβ promoter (P-NeoR-scs′) and the human Eδ enhancer (E). In addition, the constructs contain the Drosophila insulator element scs′ to inhibit transcriptional interference originating from the integration site at the 3′ end as described in the work of Zhong and Krangel (49). The 1.6-kb MINE fragment or the chicken β-globin insulator cHS4 was inserted in either orientation between the enhancer and promoter (constructs 3, 5, 6, 7, and 8) or upstream of the enhancer (constructs 4, 9, and 10). The constructs were transfected into the Jurkat T-cell line, and the number of G418-resistant colonies that express the reporter construct was determined by soft agar cloning (see Materials and Methods). The activity of the parent construct E-P-neo-scs′ (construct 1) was used as a reference and normalized to 100. Results are shown with standard errors, and the number of individual experiments (n) is indicated.