Stowe, Cassandra;
(2018)
Development of Firefly Luciferase Bioluminescence for In vivo Optical Imaging.
Doctoral thesis (Ph.D), UCL (University College London).
Abstract
Firefly luciferase is ubiquitously used as a genetic reporter for the non-invasive bioluminescence imaging of small animal models. This widespread use of Firefly luciferase in vivo has been facilitated by genetic engineering producing mutants which are extremely stable at physiological conditions. In addition, the red-shifting of bioluminescence has resulted in the enhanced penetration of light emission through biological tissue. However, the use of bioluminescence in vivo is still largely limited to the tracking of single events within a model. This is due to the differential attenuation of light <600nm, making the spectral unmixing of bioluminescent signals extremely challenging. Consequently, there is a real need to move bioluminescence into the near-infrared for dual-colour imaging. As we seem to have reached the limits of mutational based red-shifting, research has more recently focused upon chemical modification of the D-luciferin substrate. But any modification of the DLuciferin substrate is inevitably going to require subsequent mutagenesis of Firefly luciferase to optimise the light emitting reaction. The first part of this project describes the development and validation of a high throughput screening platform for bioluminescent proteins, to advance the identification of mutants with enhanced characteristics. Focus then turns to the use of genetically engineered Firefly luciferase colour mutants for in vivo bioluminescence imaging. Small animal tumour models, representing increasing tissue depth, were engrafted with Firefly luciferase colour mutants to explore the feasibility of dual colour imaging and establish the true benefit of red-shifting bioluminescence. Finally, bioluminescence in the near infra-red is used for dual bioluminescence imaging, tracking two tumour populations in a B-cell lymphoma mouse model through the spectral unmixing of Firefly luciferase colour mutants with the novel substrate infra-luciferin.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Development of Firefly Luciferase Bioluminescence for In vivo Optical Imaging |
| Event: | UCL (University College London) |
| Language: | English |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL GOS Institute of Child Health UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL GOS Institute of Child Health > Developmental Biology and Cancer Dept |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10041771 |
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