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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Nature Publishing Group
2017
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| Acceso en línea: | Registro en Scopus DOI Handle Registro en la Biblioteca Digital |
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| LEADER | 15099caa a22015977a 4500 | ||
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| 001 | PAPER-17666 | ||
| 003 | AR-BaUEN | ||
| 005 | 20230518204901.0 | ||
| 008 | 190410s2017 xx ||||fo|||| 00| 0 eng|d | ||
| 024 | 7 | |2 scopus |a 2-s2.0-85026329378 | |
| 024 | 7 | |2 cas |a DNA, 9007-49-2; DNA; Transcription Factors | |
| 040 | |a Scopus |b spa |c AR-BaUEN |d AR-BaUEN | ||
| 100 | 1 | |a Zhao, Z.W. | |
| 245 | 1 | 0 | |a Quantifying transcription factor-DNA binding in single cells in vivo with photoactivatable fluorescence correlation spectroscopy |
| 260 | |b Nature Publishing Group |c 2017 | ||
| 270 | 1 | 0 | |m Plachta, N.; Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR)Singapore; email: plachtan@imcb.a-star.edu.sg |
| 506 | |2 openaire |e Política editorial | ||
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| 520 | 3 | |a 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. |l eng | |
| 536 | |a Detalles de la financiación: Agency for Science, Technology and Research | ||
| 536 | |a Detalles de la financiación: EMBO | ||
| 536 | |a Detalles de la financiación: acknoWleDGMents We thank J. Silva for help with embryo isolation and microinjection, and V. Levi for advice on modeling. This work was supported by an A*STAR National Science Scholarship (to Z.W.Z.), a Human Frontiers Science Program Fellowship (to J.Z.), and an A*STAR Investigatorship and EMBO Young Investigator grants (to N.P.). | ||
| 593 | |a Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore, Singapore | ||
| 593 | |a Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina | ||
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