This thesis focuses on the design, construction and stable integration in mammalian cells of a natural microRNA-based genetic oscillator. This will help both in better understanding the rules underlying the periodic expression of genes observed in major biological processes, such as the circadian clock and cell-cycle, as well as, in generating new tools to probe and investigate the function of a gene in a cell, by allowing not only its over-expression or knock-down, but also its cyclic expression. The circuit involves a positive feedback loop, consisting of a transcription factor (TF) activating itself, and a negative feedback loop, using a natural micro RNA controlled by the TF, which induces degradation of the TF itself. The circuit was built in a modular way, and implemented it in two lentiviral vectors able to infect both dividing and non-dividing cells, hence suitable for many different applications. Since obtaining stable oscillations is non-trivial, a modified version of the oscillator was engineered, including an intermediate step between the TF and the microRNA, to increase the delay in the negative feedback loop. The oscillatory behavior was tested via in vivo time-lapse fluorescence microscopy in both versions of the oscillator, since both the TF(s) and the microRNA are expressed together with fluorescent reporters.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:552785 |
| Date | January 2011 |
| Creators | Siciliano, Velia |
| Publisher | Open University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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