Final Activity Report Summary - SIRNA-HCS (Study of the Molecular Mechanisms of Cell Migration by using High Content Screening and siRNA technology.) High Content Analysis (HCA) is a revolutionary technology, which combines automated fluorescence microscopy with multi-parametric analysis. The aim of this project was to use this technology in combination with small inhibitory RNA (siRNA) libraries to identify targets to inhibit T cell migration. This work should therefore identify and verify new drug-able targets involved in inflammation and metastasis with the potential for tissue specificity. A pharmacological inhibitor-based screening strategy was set up as a proof of principle approach in order to adapt the T cell migration assay to HCA, to develop protocols for analysis of the cytoskeletal rearrangement reflective of T cell migration and to automate the identification and quantification of changes in cytoskeletal shape following modulation of cell migration. Changes in the cytoskeletal shape were identified by staining with the actin label, phalloidin. Multiple analysis algorithms which described cell shape were tested It was concluded that a combination of these indices is a more sensitive and accurate reflection of changes in T cell morphology following inhibitor treatment and that this approach would be suitable for screening with siRNA libraries. We then set out to optimise delivery of siRNA/gene knockdown, acquirelibraries and put information management systems in place. The screening of 2 complete libraries (Kinome and G-protein coupled receptor - >1100 genes) was completed (intriplicate). This was followed by a secondary screen (80 genes) and a further in depth validation of 10 genes. Of these 10 genes, functional analysis of a role in cell migration was performed on 5. In parallel, we developed an analysis package particularly suited to the needs of the screening of lymphocyte migration using siRNA but which is suitable for using in other screens -HiTS http://groups.google.com/group/HiTSusers. This allowed us to speed up data management and data analysis tasks. Overall, this programme of research has allowed us to identify novel genes involved in the process of lymphocyte whiel training 4 fellows in HCA and siRNA technologies. The identification of potential druggable targets will help in the design of novel anti-inflammatory therapeutic strategies.
