Quantifying transcription factor-DNA binding in single cells in vivo with photoactivatable fluorescence correlation spectroscopy
| Autor: | Yanina D. Álvarez, Melanie D. White, Stephanie Bissiere, Nicolas Plachta, Ziqing W. Zhao, Jennifer Zenker |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Time Factors Otras Ciencias Biológicas Cell Fluorescence correlation spectroscopy Plasma protein binding Biology General Biochemistry Genetics and Molecular Biology Mammalian embryos Ciencias Biológicas Mice 03 medical and health sciences chemistry.chemical_compound In vivo medicine Animals Transcription factor Embryo culture DNA Embryo Mammalian Fluorescence Cell biology Spectrometry Fluorescence 030104 developmental biology medicine.anatomical_structure chemistry Single-Cell Analysis CIENCIAS NATURALES Y EXACTAS Protein Binding Transcription Factors |
| Popis: | 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. Fil: Zhao, Ziqing Winston. Institute of Molecular and Cell Biology; Singapur Fil: White, Melanie D.. Institute of Molecular and Cell Biology; Singapur 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 Fil: Zenker, Jennifer. Institute of Molecular and Cell Biology; Singapur Fil: Bissiere, Stephanie. Institute of Molecular and Cell Biology; Singapur Fil: Plachta, Nicolas. Institute of Molecular and Cell Biology; Singapur |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|