RNAi in Drosophila S2 Cells as a Tool for Studying Cell Cycle Progression
Genetic studies on model organisms, particularly yeasts and Drosophila melanogaster, have proven powerful in identifying the cell cycle machinery and its regulatory mechanisms. In more recent years RNAi has been used in a variety of genome-wide screens and single molecule studies to elucidate the mechanisms of cell cycle progression. In Drosophila cultured cells, RNAi is extremely simple, and a strong effect can be observed by adding the dsRNA to the cultured cells, with few complications of off-target effects. Functions in cell cycle progression can be followed by a variety of assays. One of the advantages of these cells is that they allow high-resolution spatiotemporal observations to be made by microscopy, with no particular complexity in terms of media and temperature. Here we discuss protocols for RNAi in Drosophila S2 culture cells, followed by the study of mitotic progression, through immunocytochemistry, live imaging, and flow cytometry analysis.
Key wordsMitosis cell cycle RNAi Drosophila genomewide
We would like to thank the fly community for all the available and shared reagents that were cited here. MBD and GG would like to acknowledge the Glover, the Vale, and the MBD labs for help in developing protocols and Inês Bento, Joana Lamego, Ana Rodrigues Martins, and Zita Carvalho Santos for critical reading of this chapter. Work in the MBD lab is sponsored by an EMBO Installation Grant, Fundação Calouste Gulbenkian, Fundação para a Ciência e Tecnologia, Crioestaminal, and Oeiras City Council. Work in the GG lab is sponsored by Special Coordination Funds for Promoting Science and Technology commissioned by the MEXT of Japan.
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