Masters Thesis

Advancing molecular tools for signal transduction studies in mammalian cells

Despite their fundamental importance, the dynamics of signaling pathways in living cells remain challenging to study, due to the lack of non-invasive tools for temporal assessment of signal transduction in desired cell models. Here we present a dual-reporter strategy that enables researchers to monitor signal transductions in mammalian cells in real-time, both temporally and quantitatively. This is achieved by co-expressing green fluorescent protein (GFP) and firefly luciferase (FLuc) in response to signaling stimuli. To display the versatility of this approach, we constructed and assessed eight unique pathway reporters. To further validate and demonstrate the usefulness of this system, we established stable NFкB pathway reporter cell lines. Using these stable cell lines, we monitored the activity of NFкB-mediated inflammatory pathway in real-time, both visually and quantitatively. In addition, we provide evidence that the dual reporter system is readily amenable to a high-throughput format and compatible with single cell analysis. Luciferase assay has become an increasingly important technique to monitor a wide range of biological processes in vivo and in vitro. However, the mainstay protocols require an expensive luminometer to acquire and process data, therefore limiting luciferase applications in biological research. To overcome this limitation, we have developed an alternative protocol that utilizes a commonly available cooled charge-coupled device (CCCD) instead of a luminometer for data acquisition and processing. Using a CCCD-based approach, we characterized the substrate specificity, assay linearity, signal to noise levels, and detection of fold-changes of different luciferases (Firefly, Renillar, and Gaussia). We also defined the assay parameters that are critical for the appropriate use of CCCD for the different luciferases. To demonstrate the usefulness of the proposed method in cultured mammalian cells, we conducted a case study to examine NFкB gene activation during inflammatory signaling pathway in human embryonic cells (HEK293 cells). Data collected via CCCD camera are found to be equivalent to those acquired by a luminometer, thus validating the assay protocol. By comparison, the CCCD-based protocol is more readily amenable to high-throughput applications and offers fast simultaneous data acquisition and alternative image presentation. The findings presented here demonstrate the power of the dual reporter system, significantly improving the capacity to study signal transduction pathways in mammalian cells. In addition, the CCCD-based protocol provides an important alternative for monitoring luciferase reporters. Because CCCD-based imaging technology is widely available, this approach will enable more researchers to use luciferase for monitoring biological processes.

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