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Title: Using the RISCI genetic screening platform for elucidating apoptosis signalling network
Author: Lin, Kuan Chee Bevan
ISNI:       0000 0004 2741 9311
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2013
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Considerable development in the field of nanotechnology is increasingly yielding novel applications of nanoparticles. The unique properties of nanoparticles in particular their high aspect ratio (length : width ratio), however could pose potential risks to the user. A high throughput genetic screening platform, RISCI (robotic single cDNA investigation), was previously established for the systematic evaluation of single gene activities. Here, RISCI was utilised to identify pro-apoptotic genes as well as genes involved in the positive and negative regulation of silica nanoparticle-induced cell death. This project describes the further development of the screening platform by harnessing its capability to screen a cDNA library comprising approximately 30,000 full length, completely annotated, and sequenced human genes for novel regulators of apoptosis. It integrates an extensive skill sets and is broadly organised into three major phases: Setup, Screen and Analysis. The integration of a pro-apoptosis treatment to screen for inhibitors and sensitizers is a novel aspect of the current experimental setup, along with the low redundancy library. The extensive setup phase focused on technical aspects. The cDNA library, acquired as plasmid DNA, was transformed into a bacterial host for replication and subsequent DNA isolation. A new high-throughput process was developed encompassing the production of competent bacteria and a heat shock transformation protocol, which was subsequently transferred onto the robotic platform. In parallel, the software controlling the robots was redeveloped to allow for execution of user-defined protocols while novel transfection protocols were adapted for automation. The screen identified 699 apoptosis inducers, 1,141 inhibitors and 626 sensitizers. Bioinformatics analysis revealed that the inducers were highly enriched for cell death associated terms, while the inhibitors were strongly associated with cancer profiles. Both inducers and sensitizers were predominantly achieving the functional effect on the protein level, but inhibitors were mainly transcription based. Enriched metal response genes also suggest that the silica nanoparticles were causing their toxicity through reactive oxygen species generation. Intriguingly, the screen identified many noncoding sequences as being functionally capable of regulating apoptosis. These noncoding candidates are capable of regulating the protein coding counterparts identified from the screen. The truly interesting part of the project outcome remains those unknown candidates that were implicated in apoptosis regulation for the first time. Dissemination of the consolidated candidate list would help accelerate the experimental validation of these candidates and aid other researchers in deriving novel hypotheses when the candidates are placed in their research context. [For supplementary files please contact author].
Supervisor: Grimm, Stefan ; Wharton, John Sponsor: Johnson & Johnson ; Medical Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral