Genome-wide RNAi screens in S2 cells to identify centrosome components.

Abstract

Centrosomes act as the major microtubule organizing centers in animal cells. To fully understand how the centrosome functions, a detailed analysis of its principal structural components and regulators is needed. Genome-wide RNA interference (RNAi) allows for comprehensive screening of all components. Drosophila tissue culture cells provide an attractive model for such screens. First, Drosophila centrosomes are similar to their human counterparts, but less complex. Thus, all major centrosome components are conserved and fewer redundancies apply. Second, RNAi is highly efficient in Drosophila tissue culture cells and, compared to RNAi in human cells, it is cost-effective. Finally, the availability of comprehensive libraries permits easy genome-wide screening of most of Drosophila's 14,000 protein coding genes. In this paper, we present detailed instructions for designing, performing, and analyzing a genome-wide screen in Drosophila tissue culture cells to identify centrosome components using a microscopy-based approach.