More than a decade after the completion of the human genome project, the function of a large number of genes remains to be elucidated. Forward and reverse genetic approaches have proven to be powerful tools to study gene function and have provided insights into fundamental biological processes. Furthermore, functional genetic screening can lead to a better understanding of the action of endogenous and exogenous stimuli such as hormones or drugs on biological systems. Thus far, systematic and unbiased studies have largely been limited to model organisms. However, complex disease-relevant genotypes and phenotypes cannot be studied in entirety in lower organisms creating a need for systematic approaches in human cells. This thesis describes a series of studies using forward and reverse genetic approaches combined with state-of-the-art technology in haploid human cells. The first chapter describes the development of a quantitative phenotypic read-out using a novel application of RNA-sequencing that allows the functional annotation of genes in signalling pathways. The presented data demonstrate that the employed shallow RNA-sequencing method is scalable and suitable as a read-out for reverse genetic screening. The second chapter focuses on the implementation of this method in a large reverse genetic study in human cells to functionally annotate tyrosine kinases in signalling pathways upon stimulation with a set of ten polypeptides and small molecules. The screens revealed known and unexpected interactions between different signalling molecules and pathways, validating the technical approach in a biological context. The third chapter presents a pilot study describing the set-up of a forward genetic technique for compound mode-of-action screening using a pooled human mutant cell line collection. The chemical genetic approach displayed sufficient sensitivity and allowed to monitor thousands of gene-drug interactions simultaneously. Together, this thesis combines elements to advance technological and biological aspects of functional genomics and chemical genetics.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:729871 |
| Date | January 2017 |
| Creators | Gapp, Bianca |
| Contributors | Nijman, Sebastian |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://ora.ox.ac.uk/objects/uuid:0c2ce8f8-15f3-447f-9117-8953329bd4ac |
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