Developing New Barrier Insulators for Mammalian Synthetic Biology

Synthetic gene circuits integrated into the mammalian genome are often subject to epigenetic silencing effects, such as promoter methylation, that can diminish their expression over time. As many synthetic biology applications will require the long term implementation of circuits in mammalian cells, this could represent a significant hurdle to their development. One strategy that has been devised for mitigating this problem is to flank the genes that comprise a genetic circuit with DNA elements known as Barrier insulators. Barrier insulators are a family of sequences with the ability to halt the spread of condensed heterochromatin into neighbouring euchromatic regions. In the genome, barriers are believed to exist in large numbers, delineating the boundaries of transcriptionally active domains. To date however, only a handful have been described for use as parts in mammalian expression constructs. Existing assays also make the potency of these existing insulators difficult to compare directly. Here we describe a new platform for discovering and characterizing barrier insulators in mammalian cell lines. This approach tests the capacity of a candidate barrier part to block a spreading heterochromatin domain at a defined locus. Its amenability for scaling up should enable higher throughput testing of predicted barriers and the development of improved barriers for sustained gene expression in different cell types.