Second-GenerationDrosophilaChemical Tags: Sensitivity, Versatility, and Speed

• DOI: 10.1534/genetics.116.199281
• Publication Date: 2017-02-17T01:25:34Z
• Authors: Ben Sutcliffe; Julian Ng; Thomas O Auer; Mathias Pasche; Richard Benton; Gregory S X E Jef...; Sebastian Cachero
• Keywords: Repressor lexA

Abstract Thick tissue specimens present major challenges for labeling cells and subcellular structures in a rapid reliable manner. Sutcliffe et al. present... Labeling visualizing within thick tissues, whole organs, even intact animals is key to studying biological processes. This particularly true studies of neural circuits where neurons form submicron synapses but have arbors that may span millimeters length. Traditionally, achieved by immunofluorescence; however, diffusion antibody molecules (>100 kDa) slow often results uneven with very poor penetration into the center specimens; these limitations can be partially addressed extending staining protocols over week (Drosophila brain) months (mice). Recently, we developed an alternative approach using genetically encoded chemical tags CLIP, SNAP, Halo, TMP labeling; this resulted >100-fold increase speed both mice Drosophila, at expense considerable drop absolute sensitivity when compared optimized immunofluorescence staining. We now second generation UAS- LexA-responsive CLIPf, SNAPf, Halo reagents flies. These multimerized tags, translational enhancers, display up 64-fold first-generation reagents. In addition, suite conditional reporters (4xSNAPf tag CLIPf-SNAPf-Halo2) are activated DNA recombinase Bxb1. Our new used weak strong GAL4 LexA drivers enable stochastic, intersectional, multicolor Brainbow labeling. improvements experimental versatility, while still retaining substantial advantage signature labeling, should significantly scope technology.