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Hypoxia-Inducible Factor


Hypoxia-Inducible Factor (HIF)-1 is a dimeric protein complex that plays a central role in the response to low oxygen concentrations, or hypoxia, and is a crucial physiological regulator of homeostasis, vascularization, and anaerobic metabolism. HIF is a transcription factor regulating gene expression by binding to their DNA recognition site on the target genes. Furthermore, HIF-1 has been widely studied because of its perceived therapeutic potential.  


HIF-1 allows survival and proliferation of cancerous cells due to its angiogenic properties, and the inhibition potentially could prevent the spread of cancer. With a growing understanding of the HIF-1 pathway, it has become an attractive goal to analyze the inhibition and stimulation of HIF transcriptional activity via small molecules.


The cell line was established by transfection using a pTA-HIF-luciferase reporter vector, which contains 4 repeats of HIF binding sites, a minimal promoter upstream of the firefly luciferase coding region, along with hygromycin expression vector followed by hygromycin selection. The hygromycin resistant clones were subsequently screened for CoCl2-induced luciferase activity.

TF luciferase reporter stable cell line utilizes artificial promoter constructs to drive luciferase expression.  The promoter region can consists of multiple repeats of a cis-element TF binding site, a DNA fragment from the promoter region of a known TF downstream gene, or a DNA fragment containing putative/known TF binding sites.  There are several ways that a TF can be activated, such as through extracellular stimuli or through intracellular signaling pathways.  Once activated, the TF translocates to the nucleus and often interacts with relevant co-factors to drive gene expression.  Once luciferase is expressed, it can generate light in an enzymatic assay and the amount of light measured is positively correlated with the level of TF activation.



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