Second-Generation Drosophila Chemical Tags: Sensitivity, Versatility, and Speed.

Détails

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Etat: Public
Version: Final published version
ID Serval
serval:BIB_839034DE814D
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Titre
Second-Generation Drosophila Chemical Tags: Sensitivity, Versatility, and Speed.
Périodique
Genetics
Auteur(s)
Sutcliffe B., Ng J., Auer T.O., Pasche M., Benton R., Jefferis G.S., Cachero S.
ISSN
1943-2631 (Electronic)
ISSN-L
0016-6731
Statut éditorial
Publié
Date de publication
04/2017
Peer-reviewed
Oui
Volume
205
Numéro
4
Pages
1399-1408
Langue
anglais
Notes
Publication types: Journal Article
Publication Status: ppublish
Résumé
Labeling and visualizing cells and subcellular structures within thick tissues, whole organs, and even intact animals is key to studying biological processes. This is particularly true for studies of neural circuits where neurons form submicron synapses but have arbors that may span millimeters in length. Traditionally, labeling is achieved by immunofluorescence; however, diffusion of antibody molecules (>100 kDa) is slow and often results in uneven labeling with very poor penetration into the center of thick specimens; these limitations can be partially addressed by extending staining protocols to over a week (Drosophila brain) and months (mice). Recently, we developed an alternative approach using genetically encoded chemical tags CLIP, SNAP, Halo, and TMP for tissue labeling; this resulted in >100-fold increase in labeling speed in both mice and Drosophila, at the expense of a considerable drop in absolute sensitivity when compared to optimized immunofluorescence staining. We now present a second generation of UAS- and LexA-responsive CLIPf, SNAPf, and Halo chemical labeling reagents for flies. These multimerized tags, with translational enhancers, display up to 64-fold increase in sensitivity over first-generation reagents. In addition, we developed a suite of conditional reporters (4xSNAPf tag and CLIPf-SNAPf-Halo2) that are activated by the DNA recombinase Bxb1. Our new reporters can be used with weak and strong GAL4 and LexA drivers and enable stochastic, intersectional, and multicolor Brainbow labeling. These improvements in sensitivity and experimental versatility, while still retaining the substantial speed advantage that is a signature of chemical labeling, should significantly increase the scope of this technology.

Mots-clé
Alkyl and Aryl Transferases/genetics, Alkyl and Aryl Transferases/metabolism, Animals, Brain/cytology, Brain/metabolism, Drosophila/cytology, Drosophila/genetics, Drosophila/metabolism, Drosophila Proteins/genetics, Drosophila Proteins/metabolism, Fluorescent Dyes/chemistry, Microscopy, Fluorescence/methods, Optical Imaging/methods, Recombinant Proteins/genetics, Recombinant Proteins/metabolism, Sensitivity and Specificity, Staining and Labeling/methods
Pubmed
Web of science
Open Access
Oui
Création de la notice
17/02/2017 15:31
Dernière modification de la notice
20/08/2019 14:43
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