Single site-specific integration targeting coupled with embryonic stem cell differentiation provides a high-throughput alternative to in vivo enhancer analyses
Single site-specific integration targeting coupled with embryonic stem cell differentiation provides a high-throughput alternative to in vivo enhancer analyses
复制标题
单位点特异性整合靶向与胚胎干细胞分化相结合，为体内增强子分析提供了高通量替代方案
作者：
A. Wilkinson;D. Goode;Yi;D. Dickel;Samuel D. Foster;T. Sendall;M. R. Tijssen;M. Sánchez;L. Pennacchio;A. Kirkpatrick;B. Göttgens
全面分析顺式调控元件是理解控制胚胎发育的动态基因调控网络的关键。虽然转基因动物代表了金标准试验，但它们的产生成本很高，需要大量的动物使用，并且在子宫内发育使时间过程研究复杂化。作为一种替代方法，胚胎干细胞可以很容易地在一个与胚胎发育密切相关的过程中分化。在这里，我们描述了一个高效的增强子检测平台，使用Hsp68/Venus报告盒，靶向小鼠胚胎干细胞中的Hprt位点。该平台结合了Gateway®克隆的灵活性，荧光报告细胞的活细胞可追踪性，低背景和单个拷贝插入到定义的基因组位点的优势。我们展示了两种心脏和两种造血增强剂的组织特异性增强剂活性的成功再现。此外，我们使用该分析分析了Scl+19增强子中高度保守的Ets/Ets/Gata基序的功能，结果表明Gata基序不是启动增强子活性所必需的。我们使用Gata2−/−Scl+19转基因胚胎进一步证实，Scl+19增强子的内皮活性不需要Gata2。因此，我们建立了一个有价值的工具箱来研究具有广泛适用性的基因调控网络。
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.
影响因子：
5.3
作者：
KELLER, G;KENNEDY, M;WILES, MV
通讯作者：
WILES, MV
对患有与睑裂、上睑下垂、内眦赘皮综合征 (BPES) 相关的易位 (3;21) 患者进行分子细胞遗传学评估。
DOI：
10.1006/geno.2000.6157
发表时间：
2000
期刊：
Genomics.
影响因子：
--
作者：
Praphanphoj,V;Goodman,BK;Thomas,GH;Niel,KM;Toomes,C;Dixon,MJ;Geraghty,MT
通讯作者：
Geraghty,MT
影响因子：
20.3
作者：
Lancrin, Christophe;Mazan, Milena;Lacaud, Georges
通讯作者：
Lacaud, Georges