Quantifying transcription factor-DNA binding in single cells in vivo with photoactivatable fluorescence correlation spectroscopy

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 p...

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Autores principales:	Zhao, Z.W., White, M.D., Alvarez, Y.D., Zenker, J., Bissiere, S., Plachta, N.
Formato:	JOUR
Materias:	DNA fluorescent dye transcription factor protein binding animal cell animal experiment animal tissue Article clinical protocol DNA binding embryo embryo culture embryo development female fluorescence correlation spectroscopy in vivo study information processing mouse nonhuman photoactivation priority journal process optimization protein DNA binding protein protein interaction quantitative analysis single cell analysis animal mammalian embryo metabolism procedures spectrofluorometry time factor Animals Embryo, Mammalian Mice Protein Binding Single-Cell Analysis Spectrometry, Fluorescence Time Factors Transcription Factors
Acceso en línea:	http://hdl.handle.net/20.500.12110/paper_17542189_v12_n7_p1458_Zhao
Aporte de:	Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) de Universidad de Buenos Aires

id	todo:paper_17542189_v12_n7_p1458_Zhao
record_format	dspace
spelling	todo:paper_17542189_v12_n7_p1458_Zhao2023-10-03T16:32:31Z Quantifying transcription factor-DNA binding in single cells in vivo with photoactivatable fluorescence correlation spectroscopy Zhao, Z.W. White, M.D. Alvarez, Y.D. Zenker, J. Bissiere, S. Plachta, N. DNA fluorescent dye transcription factor DNA protein binding transcription factor animal cell animal experiment animal tissue Article clinical protocol DNA binding embryo embryo culture embryo development female fluorescence correlation spectroscopy in vivo study information processing mouse nonhuman photoactivation priority journal process optimization protein DNA binding protein protein interaction quantitative analysis single cell analysis animal mammalian embryo metabolism procedures single cell analysis spectrofluorometry time factor Animals DNA Embryo, Mammalian Mice Protein Binding Single-Cell Analysis Spectrometry, Fluorescence Time Factors Transcription Factors 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. © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. JOUR info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/2.5/ar http://hdl.handle.net/20.500.12110/paper_17542189_v12_n7_p1458_Zhao
institution	Universidad de Buenos Aires
institution_str	I-28
repository_str	R-134
collection	Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA)
topic	DNA fluorescent dye transcription factor DNA protein binding transcription factor animal cell animal experiment animal tissue Article clinical protocol DNA binding embryo embryo culture embryo development female fluorescence correlation spectroscopy in vivo study information processing mouse nonhuman photoactivation priority journal process optimization protein DNA binding protein protein interaction quantitative analysis single cell analysis animal mammalian embryo metabolism procedures single cell analysis spectrofluorometry time factor Animals DNA Embryo, Mammalian Mice Protein Binding Single-Cell Analysis Spectrometry, Fluorescence Time Factors Transcription Factors
spellingShingle	DNA fluorescent dye transcription factor DNA protein binding transcription factor animal cell animal experiment animal tissue Article clinical protocol DNA binding embryo embryo culture embryo development female fluorescence correlation spectroscopy in vivo study information processing mouse nonhuman photoactivation priority journal process optimization protein DNA binding protein protein interaction quantitative analysis single cell analysis animal mammalian embryo metabolism procedures single cell analysis spectrofluorometry time factor Animals DNA Embryo, Mammalian Mice Protein Binding Single-Cell Analysis Spectrometry, Fluorescence Time Factors Transcription Factors Zhao, Z.W. White, M.D. Alvarez, Y.D. Zenker, J. Bissiere, S. Plachta, N. Quantifying transcription factor-DNA binding in single cells in vivo with photoactivatable fluorescence correlation spectroscopy
topic_facet	DNA fluorescent dye transcription factor DNA protein binding transcription factor animal cell animal experiment animal tissue Article clinical protocol DNA binding embryo embryo culture embryo development female fluorescence correlation spectroscopy in vivo study information processing mouse nonhuman photoactivation priority journal process optimization protein DNA binding protein protein interaction quantitative analysis single cell analysis animal mammalian embryo metabolism procedures single cell analysis spectrofluorometry time factor Animals DNA Embryo, Mammalian Mice Protein Binding Single-Cell Analysis Spectrometry, Fluorescence Time Factors Transcription Factors
description	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. © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
format	JOUR
author	Zhao, Z.W. White, M.D. Alvarez, Y.D. Zenker, J. Bissiere, S. Plachta, N.
author_facet	Zhao, Z.W. White, M.D. Alvarez, Y.D. Zenker, J. Bissiere, S. Plachta, N.
author_sort	Zhao, Z.W.
title	Quantifying transcription factor-DNA binding in single cells in vivo with photoactivatable fluorescence correlation spectroscopy
title_short	Quantifying transcription factor-DNA binding in single cells in vivo with photoactivatable fluorescence correlation spectroscopy
title_full	Quantifying transcription factor-DNA binding in single cells in vivo with photoactivatable fluorescence correlation spectroscopy
title_fullStr	Quantifying transcription factor-DNA binding in single cells in vivo with photoactivatable fluorescence correlation spectroscopy
title_full_unstemmed	Quantifying transcription factor-DNA binding in single cells in vivo with photoactivatable fluorescence correlation spectroscopy
title_sort	quantifying transcription factor-dna binding in single cells in vivo with photoactivatable fluorescence correlation spectroscopy
url	http://hdl.handle.net/20.500.12110/paper_17542189_v12_n7_p1458_Zhao
work_keys_str_mv	AT zhaozw quantifyingtranscriptionfactordnabindinginsinglecellsinvivowithphotoactivatablefluorescencecorrelationspectroscopy AT whitemd quantifyingtranscriptionfactordnabindinginsinglecellsinvivowithphotoactivatablefluorescencecorrelationspectroscopy AT alvarezyd quantifyingtranscriptionfactordnabindinginsinglecellsinvivowithphotoactivatablefluorescencecorrelationspectroscopy AT zenkerj quantifyingtranscriptionfactordnabindinginsinglecellsinvivowithphotoactivatablefluorescencecorrelationspectroscopy AT bissieres quantifyingtranscriptionfactordnabindinginsinglecellsinvivowithphotoactivatablefluorescencecorrelationspectroscopy AT plachtan quantifyingtranscriptionfactordnabindinginsinglecellsinvivowithphotoactivatablefluorescencecorrelationspectroscopy
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Quantifying transcription factor-DNA binding in single cells in vivo with photoactivatable fluorescence correlation spectroscopy

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