Z-REX: shepherding reactive electrophiles to specific proteins expressed tissue specifically or ubiquitously, and recording the resultant functional electrophile-induced redox responses in larval fish.
Détails
ID Serval
serval:BIB_FE6B92D5DC45
Type
Article: article d'un périodique ou d'un magazine.
Sous-type
Synthèse (review): revue aussi complète que possible des connaissances sur un sujet, rédigée à partir de l'analyse exhaustive des travaux publiés.
Collection
Publications
Institution
Titre
Z-REX: shepherding reactive electrophiles to specific proteins expressed tissue specifically or ubiquitously, and recording the resultant functional electrophile-induced redox responses in larval fish.
Périodique
Nature protocols
ISSN
1750-2799 (Electronic)
ISSN-L
1750-2799
Statut éditorial
Publié
Date de publication
05/2023
Peer-reviewed
Oui
Volume
18
Numéro
5
Pages
1379-1415
Langue
anglais
Notes
Publication types: Journal Article ; Review ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
Publication Status: ppublish
Publication Status: ppublish
Résumé
This Protocol Extension describes the adaptation of an existing Protocol detailing the use of targetable reactive electrophiles and oxidants, an on-demand redox targeting toolset in cultured cells. The adaptation described here is for use of reactive electrophiles and oxidants technologies in live zebrafish embryos (Z-REX). Zebrafish embryos expressing a Halo-tagged protein of interest (POI)-either ubiquitously or tissue specifically-are treated with a HaloTag-specific small-molecule probe housing a photocaged reactive electrophile (either natural electrophiles or synthetic electrophilic drug-like fragments). The reactive electrophile is then photouncaged at a user-defined time, enabling proximity-assisted electrophile-modification of the POI. Functional and phenotypic ramifications of POI-specific modification can then be monitored, by coupling to standard downstream assays, such as click chemistry-based POI-labeling and target-occupancy quantification; immunofluorescence or live imaging; RNA-sequencing and real-time quantitative polymerase chain reaction analyses of downstream-transcript modulations. Transient expression of requisite Halo-POI in zebrafish embryos is achieved by messenger RNA injection. Procedures associated with generation of transgenic zebrafish expressing a tissue-specific Halo-POI are also described. The Z-REX experiments can be completed in <1 week using standard techniques. To successfully execute Z-REX, researchers should have basic skills in fish husbandry, imaging and pathway analysis. Experience with protein or proteome manipulation is useful. This Protocol Extension is aimed at helping chemical biologists study precision redox events in a model organism and fish biologists perform redox chemical biology.
Mots-clé
Animals, Zebrafish/metabolism, Larva/metabolism, Proteins/metabolism, Oxidation-Reduction, Oxidants/metabolism
Pubmed
Web of science
Création de la notice
11/04/2023 15:55
Dernière modification de la notice
09/12/2023 7:02