Use of dimethyloxalylglycine (DMOG)

The SmartFlare that we have chosen for this project is specific to vascular endothelial growth factor (VEGF) mRNA.

VEGF was selected as the target because it’s transcript becomes increased in a cell as part of a characterized response to hypoxia with the aid of hypoxia inducible factor-1 (HIF-1). Active HIF-1 is a heterodimer of α and β subunits, levels of HIF-1α are kept low by oxygen-dependent prolyl hydroxylases (PHDs). When a cell becomes hypoxic, PHD levels are reduced so more HIF-1α is present to dimerise with HIF-1β forming the active heterodimer that can act as the VEGF transcription factor.

Dimethyloxalylglycine (DMOG) is a PHD inhibitor, this means that it can mimic the hypoxic state of the cell to increase the levels of HIF-1α that leads to more VEGF in the cells, acting as a great positive control in our cell system (you can find plenty of in vivo and in vitro papers at PubMed).

In a quantitative PCR experiment, SK-N-AS cells that were treated for 1 or 3 days with 500nM DMOG showed a 13 and 34-fold increase of VEGF mRNA respectively

qPCR results

Given our experimental setup, we will use cells treated with DMOG as a positive control, to ensure that there is a detectable level of VEGF mRNA within the cells for the Cy3-VEGF SmartFlare.

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