Construction of recombinant adenovirus vector containing a modified gene that codes for human hypoxia-inducible factor-1α without oxygen-dependent degradation domain

Abstract

The expression of hypoxia-inducible factor-1alpha (HIF-1alpha) in heart allografts is an important mechanism in response to ischemia/reperfusion (I/R) injury which represents the single major non-immunologic factor implicated in pathogenesis of chronic graft dysfunction (CGD). Adenoviral mediated overexpression of HIF-1alpha is a useful way to investigate the molecular mechanisms of I/R injury and the cardiac function during heart transplantation. The oxygen-dependent degradation (ODD) domain of HIF-1alpha can lead to degradation of the HIF-1alpha protein in normoxia. This will be an obstacle to steady expression of HIF-1alpha in heart allograft after transduction. In this study, we obtained the coding sequence of HIF-1alpha without ODD domain (HIF-1alphaDeltaODD) through a PCR-based method, and then generated the HIF-1alphaDeltaODD-expressing adenovirus. In normoxia, adenoviral mediated expression of HIF-1alphaDeltaODD shows constitutive activity in human cardiomyocytes, and can up-regulate heme oxygenase (HO)-1 mRNA levels significantly compared with the group transduced with HIF-1alpha-expressing adenovirus. The constructed HIF-1alphaDeltaODD-expressing adenovirus can be used to transduce allografts in animal studies to investigate the mechanism of CGD and provide a useful model to study the regulation mechanisms of genes regulated by HIF-1alpha alone.