Live and fixed imaging of translation sites at single mRNA resolution in the Drosophila embryo
| Autor: | Caroline Hoppe, Daisy J. Vinter, Hilary L. Ashe |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
Male
Science (General) Embryo Nonmammalian ved/biology.organism_classification_rank.species Gene Expression In situ hybridization Biology General Biochemistry Genetics and Molecular Biology Q1-390 Model Organisms Gene expression Developmental biology Protocol Animals Single-molecule Assays RNA Messenger Model organism Molecular Biology In Situ Hybridization In Situ Hybridization Fluorescence Regulation of gene expression Messenger RNA Microscopy General Immunology and Microbiology ved/biology General Neuroscience Translation (biology) Embryo Cell Biology Single Molecule Imaging Cell biology Protein Biosynthesis Drosophila Female Single-Chain Antibodies |
| Zdroj: | STAR Protocols STAR Protocols, Vol 2, Iss 3, Pp 100812-(2021) |
| ISSN: | 2666-1667 |
| Popis: | Summary Significant regulation of gene expression is mediated at the translation level. Here, we describe protocols for imaging and analysis of translation at single mRNA resolution in both fixed and living Drosophila embryos. These protocols use the SunTag system, in which the protein of interest is visualized by inserting a peptide array that is recognized by a single chain antibody. Simultaneous detection of individual mRNAs using the MS2/MCP system or by smFISH allows translation sites to be identified and quantified. For complete information on the generation and use of this protocol, please refer to Vinter et al. (2021). Graphical abstract Highlights • Visualization of translation of single mRNAs in Drosophila using SunTag labeling • Fixed embryo analysis reveals the mRNA translation efficiency in each expressing cell • Live SunTag confocal imaging uncovers mRNA translation dynamics • Image analysis pipelines allow quantitative and temporal information to be extracted Significant regulation of gene expression is mediated at the translation level. Here, we describe protocols for imaging and analysis of translation at single mRNA resolution in both fixed and living Drosophila embryos. These protocols use the SunTag system, in which the protein of interest is visualized by inserting a peptide array that is recognized by a single chain antibody. Simultaneous detection of individual mRNAs using the MS2/MCP system or by smFISH allows translation sites to be identified and quantified. |
| Databáze: | OpenAIRE |
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