Dual transgene strategy for live visualization of chromatin and plasma membrane dynamics in murine embryonic stem cells and embryonic tissues Journal Article

   

Authors:	Nowotschin, S.; Eakin, G. S.; Hadjantonakis, A. K.
Article Title:	Dual transgene strategy for live visualization of chromatin and plasma membrane dynamics in murine embryonic stem cells and embryonic tissues
Abstract:	To simultaneously follow multiple subcellular characteristics, for example, cell position and cell morphology, in living specimens requires multiple subcellular labels. Toward this goal, we generated dual-tagged mouse embryonic stem (ES) cells constitutively expressing differentially localized, spectrally distinct, genetically encoded fluorescent protein fusions. We have used human histone H2B fusions to fluorescent proteins to mark chromatin. This provides a descriptor of cell position, division, and death. An additional descriptor of cell morphology is achieved by combining this transgene with select lipid-modified fluorescent protein fusions that mark the plasma membrane. Using this strategy, we were able to live image cellular dynamics in three dimensions over time both in cultured ES cells and in mouse embryos generated using dual-tagged ES cells. This study, therefore, presents the feasibility of applying multiple spectrally and subcellularly distinct fluorescent protein reporters for live imaging studies in ES cells and mouse embryos. Furthermore, the increasing availability of spectral variant fluorescent proteins along with the development of methods that permit improved spectral separation now facilitate multiplexing of fluorescent reporters to provide readouts of a variety of anatomical and physiological behaviors simultaneously in living specimens. © 2009 Wiley-Liss, Inc.
Keywords:	controlled study; confocal; nonhuman; animal cell; mouse; animals; mice; animal tissue; cell death; cell division; cell function; cell structure; gene expression; embryo; embryonic stem cell; gfp; green fluorescent protein; molecular dynamics; cell line; embryo development; cell differentiation; transfection; time factors; luminescent proteins; chimera; hybrid protein; recombinant fusion proteins; feasibility studies; chromatin; cell membrane; protein transport; transgene; murinae; reporter gene; cellular distribution; embryonic stem cells; green fluorescent proteins; embryo, mammalian; plasmids; microscopy, fluorescence; cell aggregation; histone h2b; cell separation; genetic code; histones; transgenes; rfp; live imaging; mcherry; embryo culture; 3d time-lapse; fluorescent proteins; mouse embryo; membrane lipid; chromatin structure; embryonal tissue
Journal Title:	Genesis
Volume:	47
Issue:	5
ISSN:	1526-954X
Publisher:	Wiley Blackwell
Date Published:	2009-05-01
Start Page:	330
End Page:	336
Language:	English
DOI:	10.1002/dvg.20500
PUBMED:	19358158
PROVIDER:	scopus
PMCID:	PMC2875877
DOI/URL:	http://www.scopus.com/inward/record.url?eid=2-s2.0-66149101362&partnerID=40&md5=8c6ca24713765ab79e97414bc79d5321
Notes:	--- - "Cited By (since 1996): 7" - "Export Date: 30 November 2010" - "CODEN: GNESF" - "Source: Scopus"

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