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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. © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

Registro:

Documento: Artículo
Título:Quantifying transcription factor-DNA binding in single cells in vivo with photoactivatable fluorescence correlation spectroscopy
Autor:Zhao, Z.W.; White, M.D.; Alvarez, Y.D.; Zenker, J.; Bissiere, S.; Plachta, N.
Filiación:Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore, Singapore
Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina
Palabras clave: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
Año:2017
Volumen:12
Número:7
Página de inicio:1458
Página de fin:1471
DOI: http://dx.doi.org/10.1038/nprot.2017.051
Título revista:Nature Protocols
Título revista abreviado:Nat. Protoc.
ISSN:17542189
CAS:DNA, 9007-49-2; DNA; Transcription Factors
Registro:https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_17542189_v12_n7_p1458_Zhao

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Citas:

---------- APA ----------
Zhao, Z.W., White, M.D., Alvarez, Y.D., Zenker, J., Bissiere, S. & Plachta, N. (2017) . Quantifying transcription factor-DNA binding in single cells in vivo with photoactivatable fluorescence correlation spectroscopy. Nature Protocols, 12(7), 1458-1471.
http://dx.doi.org/10.1038/nprot.2017.051
---------- CHICAGO ----------
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" . Nature Protocols 12, no. 7 (2017) : 1458-1471.
http://dx.doi.org/10.1038/nprot.2017.051
---------- MLA ----------
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" . Nature Protocols, vol. 12, no. 7, 2017, pp. 1458-1471.
http://dx.doi.org/10.1038/nprot.2017.051
---------- VANCOUVER ----------
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. Nat. Protoc. 2017;12(7):1458-1471.
http://dx.doi.org/10.1038/nprot.2017.051