High throughput high content reverse genetics visual screens of ciliogenesis and cilia maintenance

摘要

Cilia are small, hair-like structures occurring on the apicalsurface of most of vertebrate cells. Defects in cilia cause arange of developmental phenotypes grouped into conditionscalled ciliopathies. Our aim is to dissect the structureand function of cilia and signalling pathways mediated bythis organelle. To evaluate this, we are performing a highthroughputsiRNA screen using siRNA pools (from theDharmacon mouse genome siRNA library) targeting over19,000 separate transcripts and immunofluorescence stainingof ciliated mIMCD3 (transformed mouse inner medullarycollecting duct) cells to determine cilia number, lengthand morphology. Secondary datasets from the screen willinclude measurements of cell size and morphology, nuclearmorphology and cell cycle profiles. We have successfullyset up a facility for high-throughput high-content imaging,optimized a reverse transfection protocol and validated aseries of positive and negative controls. We are currentlycompleting the analysis of candidate hits and expect toobtain several hundred positive hits from the whole screen.We will present the first dataset from this screen with adiscussion of prioritization strategies for the validation ofthe most relevant and interesting candidate hits.