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

摘要: Comprehensive analysis of cis-regulatory elements is key to understanding the dynamic gene regulatory networks that control embryonic development. While transgenic animals represent gold standard assay, their generation costly, entails significant animal usage, and in utero development complicates time-course studies. As an alternative, stem (ES) cells can readily be differentiated a process correlates well with developing embryos. Here, we describe highly effective platform for enhancer assays using Hsp68/Venus reporter cassette targets Hprt locus mouse ES cells. This combines flexibility Gateway® cloning, live cell trackability fluorescent reporter, low background advantages single copy insertion into defined genomic locus. We demonstrate successful recapitulation tissue-specific activity two cardiac haematopoietic enhancers. In addition, used this assay dissect functionality conserved Ets/Ets/Gata motif Scl+19 enhancer, which revealed Gata not required initiation activity. further confirmed Gata2 endothelial Gata2−/− have therefore established valuable toolbox study broad applicability.