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dc.contributor.author
Zhao, Ziqing Winston
dc.contributor.author
White, Melanie D.
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Alvarez, Yanina Daniela
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Zenker, Jennifer
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Bissiere, Stephanie
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Plachta, Nicolas
dc.date.available
2018-08-03T14:22:54Z
dc.date.issued
2017-07
dc.identifier.citation
Zhao, Ziqing Winston; White, Melanie D.; Alvarez, Yanina Daniela; Zenker, Jennifer; Bissiere, Stephanie; et al.; Quantifying transcription factor-DNA binding in single cells in vivo with photoactivatable fluorescence correlation spectroscopy; Nature Publishing Group; Nature protocols; 12; 7; 7-2017; 1458-1471
dc.identifier.issn
1750-2799
dc.identifier.uri
http://hdl.handle.net/11336/54040
dc.description.abstract
Probing transcription factor (TF)-DNA interactions remains challenging in complex in vivo systems such as mammalian embryos, especially when TF copy numbers and fluorescence background are high. To address this difficulty, fluorescence correlation spectroscopy (FCS) can be combined with the use of photoactivatable fluorescent proteins to achieve selective photoactivation of a subset of tagged TF molecules. This approach, termed paFCS, enables FCS measurements within single cell nuclei inside live embryos, and obtains autocorrelation data of a quality previously only attainable in simpler in vitro cell culture systems. Here, we present a protocol demonstrating the applicability of paFCS in developing mouse embryos by outlining its implementation on a commercial laser-scanning microscope. We also provide procedures for optimizing the photoactivation and acquisition parameters and determining key parameters describing TF-DNA binding. The entire procedure can be performed within ∼2 d (excluding embryo culture time), although the acquisition of each paFCS data set takes only ∼10 min. This protocol can be used to noninvasively reveal cell-to-cell variation in TF dynamics, as well as critical, fate-predicting changes over the course of early embryonic development.
dc.format
application/pdf
dc.language.iso
eng
dc.publisher
Nature Publishing Group
dc.rights
info:eu-repo/semantics/openAccess
dc.rights.uri
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.subject
Transcription Factor
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Mammalian Embryos
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Fluorescence Correlation Spectroscopy
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Otras Ciencias Biológicas
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Ciencias Biológicas
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CIENCIAS NATURALES Y EXACTAS
dc.title
Quantifying transcription factor-DNA binding in single cells in vivo with photoactivatable fluorescence correlation spectroscopy
dc.type
info:eu-repo/semantics/article
dc.type
info:ar-repo/semantics/artículo
dc.type
info:eu-repo/semantics/publishedVersion
dc.date.updated
2018-07-11T15:04:07Z
dc.journal.volume
12
dc.journal.number
7
dc.journal.pagination
1458-1471
dc.journal.pais
Reino Unido
dc.journal.ciudad
Londres
dc.description.fil
Fil: Zhao, Ziqing Winston. Institute of Molecular and Cell Biology; Singapur
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Fil: White, Melanie D.. Institute of Molecular and Cell Biology; Singapur
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Fil: Alvarez, Yanina Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Institute of Molecular and Cell Biology; Singapur
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Fil: Zenker, Jennifer. Institute of Molecular and Cell Biology; Singapur
dc.description.fil
Fil: Bissiere, Stephanie. Institute of Molecular and Cell Biology; Singapur
dc.description.fil
Fil: Plachta, Nicolas. Institute of Molecular and Cell Biology; Singapur
dc.journal.title
Nature protocols
dc.relation.alternativeid
info:eu-repo/semantics/altIdentifier/doi/https://dx.doi.org/10.1038/nprot.2017.051
dc.relation.alternativeid
info:eu-repo/semantics/altIdentifier/url/https://www.nature.com/articles/nprot.2017.051
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