Gene silencing of selected calcium-signalling molecules in a Drosophila cell line using double-stranded RNA interference.
Using the Drosophila melanogaster S2 cell line, stably expressing a cloned muscarinic acetylcholine receptor (AChR), DM1, we have applied gene silencing by double-stranded RNA interference (RNAi) to knock down gene products involved in DM1-mediated calcium signalling. We have shown that RNAi knock down of either the inositol 1,4,5-trisphosphate receptor (Ins(1,4,5)P(3)R), or the SERCA calcium pump in the S2-DM1 cells blocks the increase in intracellular calcium concentration ([Ca(2+)](i)) resulting from activation of the DM1 receptor by 100 microM carbamylcholine (CCh). When RNAi designed to knock down the ryanodine receptor (RyR) was tested, there was no change in the calcium increase detected in response to CCh, consistent with a failure to detect RyRs in S2-DM1 cells using RT-PCR. A combination of RNAi and calcium imaging has provided a direct demonstration of key roles for the Ins(1,4,5)P(3)R and the SERCA pump in the response to DM1 receptor activation.Thus, we show that silencing of individual genes by RNAi in a well characterised Drosophila S2 cell line offers experimental opportunities for cell-signalling studies. Future investigations with RNAi libraries taking full advantage of the wealth of new information available from sequencing the Drosophila genome, may help identify novel components of cell-signalling pathways and functionally linked gene products.
Raymond-Delpech
Valerie
V
MRC Functional Genetics Unit, Department of Human Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK.
Towers
Paula R
PR
Sattelle
David B
DB
D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects / Q000235:genetics*; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000494:pharmacology; D002460:Cell Line; D004331:Drosophila melanogaster; D020868:Gene Silencing / Q000187:drug effects / Q000502:physiology*; D034622:RNA Interference / Q000502:physiology*; D012330:RNA, Double-Stranded / Q000235:genetics* / Q000494:pharmacology; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases
D012330:RNA, Double-Stranded; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases