Date of Award

5-2011

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Life Sciences

Major Professor

Gary S. Sayler

Committee Members

Mitch Doktycz, Cynthia Peterson, Todd Reynolds, Dan Roberts

Abstract

Since its characterization, the definitive shortcoming of the bacterial luciferase (lux) bioluminescent reporter system has been its inability to express at a functional level in the eukaryotic cellular background. While recent developments have allowed for lux function in the lower eukaryote Saccharomyces cerevisiae, they have not provided for autonomous function in higher eukaryotes capable of serving as human biomedical proxies. Here it is reported for the first time that, through a process of poly-bicistronic expression of human codon-optimized lux genes, it is possible to autonomously produce a bioluminescent signal directly from mammalian cells. The low background of the bioluminescent signal, along with its characteristic lack of substrate amendment required for bioluminescent production, makes a mammalian-based lux reporter system ideal for real-time monitoring of cell culture or murine model systems. The delectability of a lux-based system provides for a functionally equivalent process to monitoring firefly luciferase-expressing cells under cell culture or subcutaneous imaging conditions without the well-documented uncertainties stemming from additional substrate introduction. However, the relatively blue-shifted emission wavelength of the lux reporter system, along with its low quantum yield, has been shown to reduce its effectiveness for use during deep tissue imaging of animal subjects. Despite these disadvantages, it has been demonstrated that a human cell line expressing the human codon-optimized lux genes can function as a biosensor for determination of human bioavailability of toxic compounds and that, by regulating the production of the luxC and luxE genes, the lux system can be employed as the first mammalian, real-time, fully autonomous bioreporter. These cell lines provide unique and efficient models for the detection and monitoring of human-relevant compounds of interest. The limiting reagent for bioluminescent production in the mammalian cellular background has been determined to be the cytosolic availability of the FMNH2 co-substrate and, in light of this evidence, directions for future optimization have been characterized and evaluated in respect to their ability to increase bioluminescent yield under these conditions.

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