Single site-specific integration targeting coupled with embryonic stem cell differentiation provides a high-throughput alternative to in vivo enhancer analyses

This is an Open Access article distributed under the terms of the Creative Commons Attribution License.-- et al.Comprehensive analysis of cis-regulatory elements is key to understanding the dynamic gene regulatory networks that control embryonic development. While transgenic animals represent the gold standard assay, their generation is costly, entails significant animal usage, and in utero development complicates time-course studies. As an alternative, embryonic stem (ES) cells can readily be differentiated in a process that correlates well with developing embryos. Here, we describe a highly effective platform for enhancer assays using an Hsp68/Venus reporter cassette that targets to the Hprt locus in mouse ES cells. This platform combines the flexibility of Gateway® cloning, live cell trackability of a fluorescent reporter, low background and the advantages of single copy insertion into a defined genomic locus. We demonstrate the successful recapitulation of tissue-specific enhancer activity for two cardiac and two haematopoietic enhancers. In addition, we used this assay to dissect the functionality of the highly conserved Ets/Ets/Gata motif in the Scl+19 enhancer, which revealed that the Gata motif is not required for initiation of enhancer activity. We further confirmed that Gata2 is not required for endothelial activity of the Scl+19 enhancer using Gata2−/− Scl+19 transgenic embryos. We have therefore established a valuable toolbox to study gene regulatory networks with broad applicability.Research in the authors' laboratory is supported by the National Centre for the Replacement, Refinement and Reduction of Animals in Research, Leukemia and Lymphoma Research, The Leukaemia and Lymphoma Society, Cancer Research UK, the Biotechnology and Biological Sciences Research Council, the Medical Research Council and core support grants from the Wellcome Trust to the Cambridge Institute for Medical Research and Wellcome Trust–MRC Cambridge Stem Cell Institute. D.E.D. was supported by the National Heart Lung and Blood Institute [grant number 5T32HL098057], and L.A.P. by the National Institute of Neurological Disorders and Stroke [grant number R01NS062859A] and by the National Human Genome Research Institute [grant numbers R01HG003988 and U54HG006997].Peer reviewe