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. 2002 Dec 10;99(Suppl 4):16433–16437. doi: 10.1073/pnas.162342499

Figure 3.

Figure 3

(A) Construction used in an assay for enhancer (ENH) blocking activity (2, 3). Expression of a gene conferring G418 resistance (neo) is driven by an erythroid-specific enhancer and promoter. This plasmid is stably transfected into K562 human erythroleukemia cells, and G418-resistant colonies are counted. Typically transfection produces tandem integrants (Control). The test for the putative insulator (I) is to insert it so that it can block enhancer action, and again to count colonies. (B) Results of inserting a 1.2-kb fragment (see Fig. 2) containing 5′HS4 on colony number in the above assay. The control has an approximately equal length of λ phage DNA on either side of the reporter to keep distances constant. The 5′HS4 element strongly reduces enhancer (ENH) activity. Other experiments show that it has a much smaller effect when placed on the other side of the enhancer, confirming the positional enhancer blocking activity (2, 3).