Abstract
Isolation of large quantities of tissue from organisms is essential for many techniques such as genome-wide screens and biochemistry. However, obtaining large quantities of tissues or cells is often the rate-limiting step when working in vivo. Here, we present a rapid method that allows the isolation of intact, single egg chambers at various developmental stages from ovaries of adult female Drosophila flies. The isolated egg chambers are amenable for a variety of procedures such as fluorescent in situ hybridization, RNA isolation, extract preparation, or immunostaining. Isolation of egg chambers from adult flies can be completed in 5 min and results, depending on the input amount of flies, in several milliliters of material. The isolated egg chambers are then further processed depending on the exact requirements of the subsequent application. We describe high-throughput in situ hybridization in 96-well plates as example application for the mass-isolated egg chambers.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Zhao T et al (2012) Growing microtubules push the oocyte nucleus to polarize the Drosophila dorsal-ventral axis. Science 336(6084):999–1003
Cai D et al (2014) Mechanical feedback through E-cadherin promotes direction sensing during collective cell migration. Cell 157(5):1146–1159
Tadros W et al (2007) SMAUG is a major regulator of maternal mRNA destabilization in Drosophila and its translation is activated by the PAN GU kinase. Dev Cell 12(1):143–155
Martin KC, Ephrussi A (2009) mRNA localization: gene expression in the spatial dimension. Cell 136(4):719–730
Ashburner M, Bergman CM (2005) Drosophila melanogaster: a case study of a model genomic sequence and its consequences. Genome Res 15(12):1661–1667
Petri WH, Wyman AR, Kafatos FC (1976) Specific protein synthesis in cellular differentiation. III. The eggshell proteins of Drosophila melanogaster and their program of synthesis. Dev Biol 49(1):185–199
Theurkauf WE, Hawley RS (1992) Meiotic spindle assembly in Drosophila females: behavior of nonexchange chromosomes and the effects of mutations in the nod kinesin-like protein. J Cell Biol 116(5):1167–1180
Jambor H et al (2015) Systematic imaging reveals features and changing localization of mRNAs in Drosophila development. Elife. doi:10.7554/eLife.05003
Lecuyer E et al (2007) Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function. Cell 131(1):174–187
Lecuyer E et al (2008) High-resolution fluorescent in situ hybridization of Drosophila embryos and tissues. CSH Protoc. 2008:pdb prot5019
Castagnetti S et al (2000) Control of oskar mRNA translation by Bruno in a novel cell-free system from Drosophila ovaries. Development 127(5):1063–1068
Ashburner M, Golic KG, Hawley RS (2005) Drosophila: a laboratory handbook, vol 1, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Starz-Gaiano M, Lehmann R (2001) Moving towards the next generation. Mech Dev 105(1-2):5–18
Acknowledgements
We would like to thank Daniel Lakatos and Franziska Friedrich for drawings of fly ovaries, Michaela Rupprecht for photos of ROMi procedure and help with processing the photos, and Daniel Lakatos for critical reading of the manuscript.
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Jambor, H., Mejstrik, P., Tomancak, P. (2016). Rapid Ovary Mass-Isolation (ROMi) to Obtain Large Quantities of Drosophila Egg Chambers for Fluorescent In Situ Hybridization. In: Dahmann, C. (eds) Drosophila. Methods in Molecular Biology, vol 1478. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6371-3_15
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6371-3_15
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6369-0
Online ISBN: 978-1-4939-6371-3
eBook Packages: Springer Protocols