I have read that GFP can be expressed from a HIF-dependent promoter so that the cells become GFP+ in hypoxia but can is it possible to encode a hydroxylation motif on the GFP protein itself in order to make it a degradation target by the VHL complex in normoxya (thus rendering the cells GFP+ in hypoxia)?
Also, have any of these systems been tried in vivo (in vivo tumor models; transgenic animals)?
Hi Pedro,
Both things can be be done and HIF- oxygen-dependent-destruction domain tagged luciferase is widely used. However, one has to consider that high level exogenous expression of ODD by itself can stabilize HIF by sequestering PHDs and VHL away from their physiological targets. On the other hand, HIF can also be stabilized at normoxia e. g. by elevated levels of ROS or certain metabolites that inhibit PHD function.
Hope that helps
Very interesting and helpful Christian, thank you.
Would you say the best option would be to have GFP regulated by a HIF-dependent promoter?
One can always try, but my personal experience is that the fluorescence level is often at or below the detection limit with promotors that work well with luciferase. Maybe you can try some superbright GFP variant or link several GFP-ORFs with T2As to have several molecules GFP produced from the same mRNA.
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