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Asymmetric mRNA localisation to specific compartments of the cell is a fundamental mechanism of -spatial and temporal regulation of gene expression. It is used by a variety of organisms and cell types to achieve different cellular functions. However, the mechanisms of mRNA localisation are not well understood. An important advance in this field has been the development of techniques that allow the visualisation of mRNA movements in living cells in real time. In this paper, we describe one approach to visualising mRNA localisation in vivo, in which RNAs containing MS2 binding sites are labelled by the MS2 coat protein fused to fluorescent reporters. We discuss the use of this mRNA-MS2/MS2CP-FP system to study mRNA localisation during Drosophila oogenesis, and provide a detailed explanation of the steps required for this approach, including the design of the mRNA-MS2 and MS2CP-FP constructs, the preparation of fly oocytes for imaging, the optimal microscope configurations for live cell imaging, and strategies for image processing and analysis.

Original publication

DOI

10.1007/978-1-61779-005-8_17

Type

Journal article

Journal

Methods Mol Biol

Publication Date

2011

Volume

714

Pages

265 - 283

Keywords

Animals, Animals, Genetically Modified, Bacteriophages, Capsid Proteins, Drosophila melanogaster, Female, Image Processing, Computer-Assisted, Luminescent Proteins, Male, Molecular Imaging, Oogenesis, RNA Transport, RNA, Messenger