Regular Article
The Sea Urchin sns Insulator Blocks CMV Enhancer following Integration in Human Cells

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Abstract

Insulators are a new class of genetic elements that attenuate enhancer function directionally. Previously, we characterized in sea urchin a 265-bp-long insulator, termed sns. To test insulator activity following stable integration in human cells, we placed sns between the CMV enhancer and a tk promoter upstream of a GFP transgene of plasmid or retroviral vectors. In contrast to controls, cells transfected or transduced with insulated constructs displayed a barely detectable fluorescence. Southern blot and PCR ruled out vector rearrangement following integration into host DNA; RNase protection confirmed the enhancer blocking activity. Finally, we demonstrate that two cis-acting sequences, previously characterized in sea urchin, are also specific binding sites for human proteins. We conclude that sns interferes with enhancer promoter interaction also in a human chromatin context. The relatively small size, evolutionary conservation and apparent lack of enhancer specificity might result useful in gene transfer experiments in human cells.

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    To whom correspondence should be addressed at Dipartimento di Biologia Cellulare e dello Sviluppo, Parco d'Orleans II, 90128 Palermo, Italy. Fax: +39091/6577430. E-mail: [email protected].

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