Development and Optimization of a Reporter Gene
Assay to Measure Activation of NF-B
Sylvia Sarponmaah1, Courtney Riley2 and Craig Woodworth3
Department of Biology, Clarkson University
NF-B is a transcription factor that alters cell behavior in important ways. NF-kB inhibits apoptosis
(programmed cell death), it increases cell proliferation, and it contributes to the development of several
types of human cancer. Human papillomaviruses are small tumor viruses that alter NF-kB activity and
contribute to cervical cancer. Our long term goal is to investigate how HPVs activate NF-B in normal
human cervical epithelial cells. To examine this question, we have developed a reporter gene assay to
measure NF-kB activity in cells cultured from biopsies of human cervix. The reporter gene contains the
NF-B promoter (the DNA sequence that regulates NF-kB-induced genes) cloned in front of the firefly
luciferase gene. The protein encoded by the luciferase gene fluoresces strongly when the NF-kB
promoter is active, making it easy to determine the level of activation based on the level of luminescence.
We also used a second reporter gene composed of the renilla luciferase gene attached to a weak thymidine
kinase promoter. This reporter is active in all cells and serves as a baseline for comparison. Both reporter
genes were introduced into cultures of human cervical epithelial cells using lipofection. Lipofection
involves coating the reporter gene DNA with lipid (liposomes), and allowing cells to take up the lipid and
DNA. The first goal of this study was to optimize the transfection protocol in such a way that high
transfection rates were achieved. This is important because too much DNA and lipid can be toxic to the
cells, whereas too little results in no reporter activity. Various combinations of DNA, Lipofectamine and
Plus Reagent were tested to determine the optimal ratio. We found that the optimal amount of DNA and
lipofectamine varied depending on the cells and the tissue culture conditions. When transfection assays
were performed on normal cells in 12 well plates, levels of DNA below 0.2 microgram and volumes of
lipofectamine below 2.0 microliter gave optimal results. After the reporter gene assay is optimized, our
next goal will be to compare NF-kB activation in normal cervical cells and cells at different stages of
cancer development (HPV-infected, HPV-immortalized and cervical cancer. We will also determine
whether HPV activates NF-kB by stimulating the epidermal growth factor receptor.
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3
Class of 2007, Biology major, Clarkson University and Ronald McNair Scholars Program - poster
Class of 2009, Biology major and Honors Program, Clarkson University
Associate Professor of Biology, Clarkson University
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