title: An embryonic stem cell-based system for rapid analysis of transcriptional enhancers.
abstract: With the growing use of genome-wide screens for cis-regulatory elements, there is a pressing need for platforms that enable fast and cost-effective experimental validation of identified hits in relevant developmental and tissue contexts. Here, we describe a murine embryonic stem cell (ESC)-based system that facilitates rapid analysis of putative transcriptional enhancers. Candidate enhancers are targeted with high efficiency to a defined genomic locus via recombinase-mediated cassette exchange. Targeted ESCs are subsequently differentiated in vitro into desired cell types, where enhancer activity is monitored by reporter gene expression. As a proof of principle, we analyzed a previously characterized, Sonic hedgehog (Shh)-dependent, V3 interneuron progenitor (pV3)-specific enhancer for the Nkx2.2 gene, and observed highly specific enhancer activity. Given the broad potential of ESCs to generate a spectrum of cell types, this system can serve as an effective platform for the characterization of gene regulatory networks controlling cell fate specification and cell function.
authors.affiliation: Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
authors.forename: Kaloyan M
authors.initials: KM
authors.lastname: Tsanov
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Nishi
authors.forename: Kevin A
authors.initials: KA
authors.lastname: Peterson
authors.forename: Jing
authors.initials: J
authors.lastname: Liu
authors.forename: Manfred
authors.initials: M
authors.lastname: Baetscher
authors.forename: Andrew P
authors.initials: AP
authors.lastname: McMahon
mesh_terms: D001483:Base Sequence; D002454:Cell Differentiation; D017931:DNA Primers; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D018390:Gene Targeting; D000090322:Homeobox Protein Nkx-2.2; D018398:Homeodomain Proteins; D006801:Humans; D007150:Immunohistochemistry; D009687:Nuclear Proteins; D014157:Transcription Factors
chemical_list: D017931:DNA Primers; D000090322:Homeobox Protein Nkx-2.2; D018398:Homeodomain Proteins; C000715631:NKX2-2 protein, human; D009687:Nuclear Proteins; D014157:Transcription Factors
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