English   español  
Por favor, use este identificador para citar o enlazar a este item: http://hdl.handle.net/10261/129179
Título

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

AutorSánchez, María José ; Pennacchio, Len A.; Kirkpatrick, Aileen M.; Göttgens, Berthold
Palabras claveES cells
Enhancer
Haematopoiesis
Transcription
Fecha de publicación2013
EditorCompany of Biologists
CitaciónBiology Open 2(11): 1229-1238 (2013)
ResumenComprehensive 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.
DescripciónThis is an Open Access article distributed under the terms of the Creative Commons Attribution License.-- et al.
Versión del editorhttp://dx.doi.org/10.1242/bio.20136296
URIhttp://hdl.handle.net/10261/129179
DOI10.1242/bio.20136296
E-ISSN2046-6390
Aparece en las colecciones: (CABD) Artículos
Ficheros en este ítem:
Fichero Descripción Tamaño Formato  
Single site-specific.pdf1,04 MBAdobe PDFVista previa
Visualizar/Abrir
Mostrar el registro completo
 

Artículos relacionados:


NOTA: Los ítems de Digital.CSIC están protegidos por copyright, con todos los derechos reservados, a menos que se indique lo contrario.