Abstract
SummaryComprehensive 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.
Highlights
The intricate process of embryonic development involves dynamic interactions of transcription factors with gene regulatory elements within gene regulatory networks (GRNs) (Davidson, 2010; Pimanda and Gottgens, 2010)
Summary Comprehensive analysis of cis-regulatory elements is key to understanding the dynamic gene regulatory networks that control embryonic development
We describe a highly effective platform for enhancer assays using an Hsp68/ Venus reporter cassette that targets to the hypoxanthine guanine phosphoribosyl transferase 1 (Hprt) locus in mouse embryonic stem (ES) cells
Summary
The intricate process of embryonic development involves dynamic interactions of transcription factors with gene regulatory elements within gene regulatory networks (GRNs) (Davidson, 2010; Pimanda and Gottgens, 2010). For the mouse model this can be expensive in animal usage since variable copy-numbers of the transgene and integration positional effects require multiple transgenic lines in order to establish reproducible expression patterns. This variability can be circumvented by exploiting embryonic stem (ES) cell lines with a defective hypoxanthine guanine phosphoribosyl transferase 1 (Hprt) gene, since strategies that restore Hprt function enable selection for single copy integration. Regardless of the procedure employed for acquiring transgenic mouse embryos their intra-uterine development complicates time-course studies
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