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Title: Generation of a murine ES cell system deficient in microRNA processing for the identification of microRNA targets
Author: Davis, M. P.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2009
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I have developed a system in mouse embryonic stem (ES) cells to simply and rapidly derive gene lists enriched for miRNA targets. I have disrupted miRNA processing by the targeted insertion of a gene trap cassette into the second allele of Dgcr8 in cell lines that already bear a gene trap within their first allele. This led to a broad reduction of miRNA processing in these cells. As a consequence of the disruption of this locus I was able to identify a number of miRNAs that appear to be processed in DGCR8 independent manner. I proceeded to transfect these cells with Es-cell-expressed miRNA mimics. I used microarrays to identify transcripts that are down regulated as a consequence of the miRNA reintroduction. By comparing transcripts that had been up regulated upon the depletion of Dgcr8 to this set I was able to create miRNA target lists for mmu-miR-25 and mmu-miR-291a-3p. These lists should be enriched for functionally relevant, co-expressed targets, moderated for miRNA mimic over expression and to a large extent devoid of interference from target saturation and combinatorial regulation. The system should also not be susceptible to problems associated with functional redundancy. In total I identified 25 target candidates for miR-291a-3p and 40 candidates for miR-25. Amongst these genes are a number of oncogenes and tumour suppressor genes in addition to genes involved in cell cycle regulation and extra-cellular signal transduction. In conclusion it appears that miRNAs play a fundamental role in the regulation of the ES cell transcriptome and as such are deserving of considerable future research. It is my belief that the method presented in this thesis could contribute significantly to this effort by providing substantial and experimentally derived miRNA candidate target lists upon which to base future hypotheses.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available