Assaying the Drosophila negative feedback loop with RNA interference in S2 cells

Transcriptional negative feedback loops play a critical role in the molecular oscillations of circadian genes and contribute to robust behavioral rhythms. In one key Drosophila loop, CLOCK and CYCLE (CLK/CYC) positively regulate transcription of period (per). The period protein (PER) then represses this transcriptional activation, giving rise to the molecular oscillations of per RNA and protein. There is evidence that links molecular oscillations with behavioral rhythms, suggesting that PER also regulates the expression of downstream genes, ultimately resulting in proper behavior rhythmicity. Phosphorylation of PER has also been shown to be critical for rhythms. Doubletime (DBT) and casein kinase II (CKII) have been implicated in the phosphorylation of PER, which affects its stability as well as nuclear localization. We investigated the role of these kinases on PER transcriptional repression using the Drosophila S2 cell line in combination with RNA interference (RNAi) to knock down specific gene expression. This article describes the methods used to study PER repression activity in the S2 cell system as well as to exploit RNAi in this system. We also include protocols for immunocytochemistry and the application of leptomycin to differentiate direct effects on repression from indirect effects on subcellular localization. Finally, we discuss the generation of stable cell lines in the S2 cell system; these will be useful for experiments requiring homogeneous cell populations.