| Popis: |
Bioluminescence imaging (BLI) of transgenic mice expressing the firefly luciferase gene luc has been used to monitor continuous changes in gene expression in cultures and in the whole animal. This dissertation describes new techniques for bioluminescence imaging of gene activity at whole-animal and cellular levels. To record gene expression at the whole-animal level, the luciferase substrate luciferin is typically injected into mice prior to imaging. To avoid the effects of handling and stress from injection on expression of the transgene, a new method for delivering luciferin orally was developed and tested. Orally administered luciferin was found to be readily absorbed from the digestive tract and produced levels of bioluminescence in the whole animal that were similar to results following injection. Imaging at the cellular level focused on identifying a new model system for analyzing circadian rhythms of vertebrate neurons. In the mammalian circadian system, multiple circadian pacemakers located throughout the body are synchronized to the external 24-hour day by a molecular circadian clock. BLI has been used to detect rhythmic expression of mPer1 and mPer2 genes in the “master” clock of the suprachiasmatic nucleus (SCN) which regulates daily physiology and behavior and also sends timing cues to peripheral oscillators outside the SCN. Because SCN neurons are small and the SCN contains many neuronal phenotypes, subcellular imaging in identifiable neurons is challenging. As a result of the limitations of the SCN, larger neurons with similar circadian and molecular properties were sought, resulting in a focus on the mesencephalic trigeminal nuclei (Me5). The Me5 are a heterogeneous group of large (30-40 mm) pseudo-unipolar primary sensory neurons and multipolar interneurons that receive proprioceptive signals from spindle organs of the masseter muscles and periodontal ligaments of the teeth. Methods for Me5 cell culture and bioluminescence imaging of Me5 organotypic explants were developed using mPer1::luc and mPer2luc transgenic mice. The period and phase of circadian rhythms in mPer1 and mPer2 gene expression in Me5 neurons were characterized. Most importantly, bioluminescence imaging and immunohistochemistry were used to provide evidence of a cell-autonomous circadian oscillator in Me5 neurons. The unique characteristics of the Me5 and its use as an alternative molecular model may provide opportunities to expand cellular studies of neural circadian pacemakers beyond the limitations of the SCN. Similarly, the ability to image bioluminescence after administering luciferin through drinking water could enable circadian rhythms in gene expressions to be monitored in various locations in animal models and more efficiently associate cellular circadian clock mechanisms with resulting animal behavior. |
