Characterization of a core region in the A2UCOE that confers effective anti-silencing activity

We have previously shown that reliability of the A2UCOE in driving transgene expression can be attributed to its resistance to DNA methylation, and its ability to confer this property to linked regulatory sequences. In order to gain a better understanding of how resistance to DNA methylation from the A2UCOE is conferred, and whether the anti-silencing effect from the A2UCOE is confined within a core region, we evaluated the anti-silencing effect of different sub-domains. We found that maximal epigenetic regulatory activity was contained within a 455 bp element derived from the CBX3 region when tested in the context of a lentiviral vector in murine embryonic stem (ES) cells and human inducible pluripotent stem (iPS) cells. This region possessed an active chromatin signature, and operated effectively in cis to protect linked heterologous regulatory elements from methylation, thereby conferring stable transgene expression. Defined UCOE elements may be particularly useful for use in vectors where gene expression is desired in methylation-prone chromatin environments such as those encountered in pluripotent stem cells.