SIRNA-HCS · Study of the molecular mechanisms of cell migration by using high content screening and siRNA technology
FP6 — Marie Curie Actions (Human Resources and Mobility)
- Duration
- 2006-06-01 → 2010-01-31
- EU contribution
- €569,289
- Participants
- 3
- Scheme
- TOK
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Results in brief
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.
Data: CORDIS, © European Union
Project objective
Cell migration is a key step for the development of inflammatory and metastatic disease. Mechanisms involved in cell migration include alterations in adhesion molecules and chemokine receptor status, activation of signalling molecules and cytoskeletal rearrangement. Cell-based high content screening (HCS) is a revolutionary technology which allows subcellular imaging of temporary cellular events as well as analysis of intracellular events, permitting the study of deeper biological information at the sub-cellular level.Hence, due to its high throughput capacity, this technology is likely to improve the bottlenecks in the process leading towards drug discovery, and the establishment of this technological platform in Europe will have immediate applications to many of the causes of morbidity and mortality within the European Union. The laboratory of Prof. Dermot Kelleher, the coordinator of this proposal, is one of the first academic institutions in Europe to acquire such a technology. In this application we propose to combine the efficiency of inhibitory small inhibitory RNA (siRNA) libraries with the high throughput capacity of cell-based HCS to the study of cell migration in the context of neoplastic diseases and inflammation.This project requests research fellows with expertise in the areas of cell biology/cell migration combined with HCS, IT/bioinformatics, molecular biologists with experience of inhibitory RNA and high throughput transfection technologies and cell biologists with industrial/drug screening experience. This transfer of knowledge to the host institution using siRNA in combination with HCS to study the molecular mechanisms of cell migration will be essential to help establish and develop this novel technology and its use to accelerate the process of drug discovery.
Original text from CORDIS.
Participants
- THE PROVOST FELLOWS AND SCHOLARS OF THE COLLEGE OF THE HOLY AND UNDIVIDED TRINITY OF QUEEN ELIZABETH NEAR DUBLIN · DUBLINCoordinatorCity levelIreland
- BENITEC LTD. · CALIFORNIACity levelUnited States
- CELLOMICS INC · PITTSBURGH PACountry levelUnited States
Links
Data: CORDIS, © European Union