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Title: RNA interferences in ES cells
Author: Ridgway, Rachel A.
ISNI:       0000 0004 2748 0243
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2005
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The aim of this project was to develop methods for conditional induction of gene specific silencing in murine embryonic stem (mES) cells using RNA interference (RNAi). RNA interference (RNAi) is a process whereby double stranded (ds) RNA can reduce the expression of a gene by selective mRNA degradation. This thesis describes the investigation of several methods of inducing RNAi in mES cells. These included design and transfection of chemically synthesised small interfering (si) RNAs the transfection of vectors expressing short hairpin (sh) RNAs and construction of a vector that expresses hairpin dsRNA under tetracycline control. A specific reduction in GFP expression was observed by the transfection of siRNAs targeting either plasmid or endogenously expressed GFP. Transfections of two out of three Oct-4 siRNAs were shown to reduce the expression of Oct-4. mES cell lines were made that stably express shRNAs targeting Rex-1 and Laminin Bl. Analysis of gene expression by semi-quantitative RT-PCR showed knockdown of the target genes in some cell lines. Transfection of another vector expressing Oct-4 shRNA was shown to induce differentiation of mES cells as measured by an increase in cell size. Methods were developed for the cloning of an inverted-repeat of target genes (Oct-4, LMNA) into a tetracycline-inducible vector. The system was tested in a HEK 293 cell line expressing the tet-responsive transactivator protein. The hairpin dsRNA was shown to be expressed when doxycycline was added to the cells but no knockdown of LMNA was observed. The use of siRNAs and shRNAs to induce gene specific silencing in ES cells was shown. Although fast and efficient methods for the assaying of RNAi induced knockdown in cells were not demonstrated. Therefore the potential of RNAi as a high throughput system for establishing gene function in mES has yet to be realised.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available