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RESEARCH PRODUCT

321. Sea Urchin sns Chromatin Insulator Prevents Silencing and Positional Effect Variegation of Oncoretroviral Vectors Transgene Expression in Murine Erythroid Cell Line

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Silencing and position effect are considered significant obstacles to obtain a consistent level of transgene expression in viral gene therapy. Furthermore recent studies had shown that retroviruses tend to land on active genes with the potential consequence of insertional mutagenesis. The inclusion of elements, such as chromatin insulators, capable to insulate a gene from the surrounding chromatin effects at the integration site should improve both efficacy and safety of gene therapy vectors. We have previously characterized a 265 bp insulator element, termed sns, localized at the 3’ end of the early histone H2A gene of the sea urchin Paracentrotus lividus. This sequence contains three cis-acting elements (Box A, Box B, and Box C+T) all needed for the enhancer blocking activity in both sea urchin and human cells. By colony assays, in human (K562) and mouse (Mel) erythroid cell lines, we have recently demonstrated that the sns insulator displays directional enhancer-blocking activity in that it interferes with the communication between the human beta-globin enhancer (LCR) and the gamma-globin promoter. By electrophoretic mobility shift assays (EMSA) we found bindings of sns insulator with the erythroid specific GATA1 and the ubiquitous Oct1, and Sp1 transcription factors.