Barriers Composed of tRNA Genes Can Complement the Benefits of a Ubiquitous Chromatin Opening Element to Enhance Transgene Expression

Random integration of transgenes into host cell genomes often occurs in epigenetically unstable regions, leading to variable and unreliable transgene expression. To address this, biomanufacturing organizations frequently employ barrier elements, such as the widely-used ubiquitous chromatin opening element (UCOE). We have compared UCOE barrier activity against a barrier provided by tRNA genes. We demonstrate that the tRNA genes provide a more effective barrier than a UCOE in preventing transgene silencing in Chinese hamster ovary (CHO) cells. Nevertheless, the UCOE offers other benefits, increasing expression strongly, albeit transiently, and reducing production variability. Both the UCOE and tRNA genes counteract the repressive heterochromatin mark H3K9me3, but only the tRNA genes sustain euchromatic H3K27ac and recruitment of RNA polymerase II (Pol II) throughout long-term culture. A hybrid combining these distinct types of elements can provide benefits of both, enhancing expression in a more enduring manner. This synthetic hybrid offers potential for biomanufacturing applications.