100 likes | 235 Views
Combinatorial RNAi screens for cell growth and viability. Co-RNAi of 84 protein and lipid phosphatases in Drosophila Map Interactions to identify phosphatases are involved in similar processes Scalability: pilot experiments for larger combinatorial screens. Types of interactions. A*B*. wt.
E N D
Combinatorial RNAi screens for cell growth and viability • Co-RNAi of 84 protein and lipid phosphatases in Drosophila • Map Interactions to identify phosphatases are involved in similar processes • Scalability: pilot experiments for larger combinatorial screens
Types of interactions A*B* wt A* B* A*B* wt A* A B viability positive (synergistic) interaction X B negative (buffering) interaction viability A B* negative (suppressing) interaction viability B A A*B* wt A* B*
Experimental set up Int Pos Neg P1 4 Plates 6 Plates P2 P3 . . . P1 P2 P3 . . . P – Neg P – Pos, P – Int Pi – Pj Pi – Pi 24 Plates
Experimental set up Ready-to-screen assay plates Seed cells (10.000 – 20.000) Transfection of dsRNAs Kc167 SL2 S2R+ 5 days incubation (ensure protein depletion) Readout (single channel)
Experimental set up • “all against all” • single knock-downs (PPX-Neg) • double knock-downs (PPX-PPX) • combination knock-downs (PPX-PPY) • possible findings • viability hits • control rescues • PPX_PPY interactions PP3 PP2 PP1 intermediate neg pos PP21 ………………………………………………………. PP1 PP2 PP3 … … … … … … … … … … … … … PP14 pos neg Plate 1 of 24
Quality assessment PPX-PPY vs PPY-PPX Slope ~1 Outliers Systematic error
Interaction estimates viability hits single effects synergistic interactions antagonistic interactions interactions
cluster of lipid phosphatases (CG11437, CG11438, wun) interact with JNK signaling Related phosphatases share interaction profiles synergistic antagonistic interactions
Networks based on synthetic interactions Limited number of PP with many interactions, e.g. puc PP1- a96 Expansion to larger network sizes, more complex phenotypes Application to RNAi-drug interactions