Identifying the cis-regulatory mechanisms controlling gene expression will be crucial to understand not only how normal cellular development proceeds but also how dysregulation of transcription causes disease.
At present, the definitive assay for attributing tissue specific function to regulatory elements is the generation of transgenic reporter mice resulting in significant use of animals. Development of alternative non-animal assays therefore offers significant scope for reducing animal use across the UK and beyond.
Here, we propose to develop reagents and protocols that will lead to a substantial replacement in the use of transgenic mice for analysing regulatory elements with in vitro based transgenic embryonic stem (ES) cell differentiation assays. Building on current ES cell targeting strategies we aim to modify and develop a toolbox of vectors that permits regulatory elements to be easily integrated into a single site in the genome. By robustly testing the activity of regulatory elements during the in vitro differentiation of ES cells and validating the cell types that can be targeted we intend to demonstrate that ES cell based assays provide an exciting and viable alternative to studying gene regulatory elements in transgenic mice. Moreover, with similar methods of targeting human ES cell lines now emerging there is further scope to replace a greater number of transgenic animals with ES cell based differentiation assays.
In addition to reducing the numbers of animals used in this type of research the proposed studies will help identify key lineage specific regulatory elements. Such elements in turn hold great potential to drive the development of novel in vitro differentiation protocols which will be vital to harness the promise of embryonic stem cells for regenerative medicine. This proposal therefore addresses important issues both in fundamental biomedical research as well as developing technologies with the potential of significant replacement of animal experiments.
Dickel DE, et al. (2014) Function-based identification of mammalian enhancers using site-specific integration. Nat Methods 11(5):566-71. doi: 10.1038/nmeth.2886.
Wilkinson AC, et al. (2013) Single site-specific integration targeting coupled with embryonic stem cell differentiation provides a high-throughput alternative to in vivo enhancer analyses. Biol Open 2(11):1229-38. doi: 10.1242/bio.20136296.
Smith AM, et al. (2012) Integration of Elf-4 into stem/progenitor and erythroid regulatory networks through locus-wide chromatin studies coupled with in vivo functional validation. Mol Cell Biol 32(4): 763-73 doi:10.1128/MCB.05745-11