Construction of recombinant adenovirus vector containing a modified gene that codes for human hypoxia-inducible factor-1alpha without oxygen-dependent degradation domain.
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.
Wei
Liang
L
Key Laboratory of Transplant Engineering and Immunology of Health Ministry of China, West China Hospital, Sichuan University, Chengdu 610041, China.
Lu
Jun
J
Feng
Li
L
Li
Shengfu
S
Shan
Juan
J
Li
Youping
Y
D000256:Adenoviridae / Q000235:genetics*; D001483:Base Sequence; D004274:DNA, Recombinant / Q000096:biosynthesis*; D005786:Gene Expression Regulation / Q000187:drug effects; D005822:Genetic Vectors / Q000096:biosynthesis*; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D032383:Myocytes, Cardiac / Q000378:metabolism; D010100:Oxygen / Q000494:pharmacology*; D010957:Plasmids / Q000235:genetics; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D014161:Transduction, Genetic
D004274:DNA, Recombinant; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger; D010100:Oxygen