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

Authors
  • Wilkinson, Adam C1
  • Goode, Debbie K
  • Cheng, Yi-Han
  • Dickel, Diane E
  • Foster, Sam
  • Sendall, Tim
  • Tijssen, Marloes R
  • Sanchez, Maria-Jose
  • Pennacchio, Len A
  • Kirkpatrick, Aileen M
  • Göttgens, Berthold
  • 1 Cambridge Institute for Medical Research and Wellcome Trust-MRC Cambridge Stem Cell Institute, University of Cambridge , Hills Road, Cambridge CB2 0XY , UK.
Type
Published Article
Journal
Biology open
Publication Date
Jan 01, 2013
Volume
2
Issue
11
Pages
1229–1238
Identifiers
DOI: 10.1242/bio.20136296
PMID: 24244860
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

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.

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