Luciferase Assay
To control the functionality of the TetR-ODD-VP16 system, the HEK 293T and Hep G2 cell lines were cotransfected using the pTET Off-ODD and pTRE-Luc vectors.
100 µM of CoCl2 was added to the cell medium to establish 1% O2 in the medium. The cells were collected 9 hours later. Luminometric measurement under Thermo Varioscan for 613 nm was done and the data of the following graph were acquired:
The observation of the operational level of the TetR-ODD-VP16 did not yield fruitful results as the cells reached out of the dish when 100 µM of CoCl2 were added to HEK 293T medium.
When the Tet Off-ODD and pTRE-Luc vectors were cotranfected to Hep G2 cells in hypoxic medium, there was a 4 times increase in Luciferase concentration in respect to normoxic medium.
In the light of these results, it is possible to say that the Tet-ODD-VP16 system was successfully synthesized and a novel hypoxia inducible system was introduced for future use in studies indulged into examining hypoxic conditions.
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