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

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

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

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

Summary 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

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

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

Function-based identification of mammalian enhancers using site-specific integration

The accurate and comprehensive identification of functional regulatory sequences in mammalian genomes remains a major challenge. Here we describe Site-specific Integration FACS-sequencing (SIF-seq), an unbiased, medium-throughput functional assay for the discovery of distant-acting enhancers. Pluripotent cell reporter assays, targeted single-copy genomic integration, and flow cytometry are coupled with high-throughput DNA sequencing to enable parallel screening of large numbers of DNA sequences. We demonstrate the utility of this method by functionally interrogating>500 kb of mouse and human sequence for enhancer activity and identifying embryonic stem (ES) cell enhancers at pluripotency loci including NANOG. We also demonstrate the effectiveness of the approach in differentiated cell populations through the identification of cardiac enhancers from cardiomyocytes and neuronal enhancers from neural progenitors. SIF-seq is a powerful and flexible method for the de novo functional identification of mammalian enhancers in a potentially wide variety of cell types. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research

Discovery-based science education: functional genomic dissection in Drosophila by undergraduate researchers.

How can you combine professional-quality research with discovery-based undergraduate education? The UCLA Undergraduate Consortium for Functional Genomics provides the answe

Genomewide Clonal Analysis of Lethal Mutations in the Drosophila melanogaster Eye: Comparison of the X Chromosome and Autosomes

Using a large consortium of undergraduate students in an organized program at the University of California, Los Angeles (UCLA), we have undertaken a functional genomic screen in the Drosophila eye. In addition to the educational value of discovery-based learning, this article presents the first comprehensive genomewide analysis of essential genes involved in eye development. The data reveal the surprising result that the X chromosome has almost twice the frequency of essential genes involved in eye development as that found on the autosomes