Fighting hypoxia in printed tissue

Hypoxia-inducible promoters

Constructs and methodology

In order to test hypoxia-dependent gene expression, we created luciferase constructs that allow the quantification of expression levels via a luciferase assay. The expression of these constructs is driven by a hypoxia-inducible promoter, which results in the translation of the luciferase and its secretion into the medium. By taking samples of the medium after 12 h and 24 h after the exposure of HEK293T cells to hypoxia and measuring luciferase activity by a corresponding assay, we are able to quantify hypoxia-dependent gene expression. In order to determine whether the gene expression is really only responsive to hypoxia - but inactive during normal oxygen concentrations - we were furthermore running a control at normal oxygen concentrations (X% oxygen).

For the assay, a total of eight constructs were tested, six of which are luciferase constructs for the quantification of expression levels and two of which are VEGF-constructs that are used to confirm the expression of the actual growth factor by our cells. Luciferase constructs all contain the hypoxia promoter, a signal peptide, a coding sequence and a polyadenylation signal, but vary in the number of hypoxia-response elements.

Of the two constructs that do not contain a luciferase, but the actual growth factors (VEGF and PDGF), one is expressed via a hypoxia-promoter (4x HRE) and one via a CMV promoter as a positive control. Expression levels are hereby determined via an ELISA.

References