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Role of the oxygen-dependent degradation domain in a hypoxia-inducible gene expression system in vascular endothelial growth factor gene therapy

Authors
 HongLian Jin  ;  Meng-Lu Liu  ;  Hyun Ah Kim  ;  Minhyung Lee  ;  SungSu An  ;  JinSoo Oh  ;  Joon Cho  ;  Seong Yi  ;  KeungNyun Kim  ;  DoHeum Yoon  ;  Yoon Ha 
Citation
 SPINE, Vol.34(26) : 952-958, 2009 
Journal Title
 SPINE 
ISSN
 0362-2436 
Issue Date
2009
MeSH
Animals ; Astrocytes/physiology ; Cells, Cultured ; Enzyme-Linked Immunosorbent Assay ; Fluorescent Antibody Technique ; Gene Expression/genetics* ; Genes, Reporter/genetics ; Genetic Therapy/methods* ; Hypoxia/genetics* ; Hypoxia/therapy ; Male ; Neurons/physiology ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Spinal Cord Injuries/genetics* ; Vascular Endothelial Growth Factor A/genetics* ; Vascular Endothelial Growth Factor A/therapeutic use*
Keywords
spinal cord injury ; hypoxia-inducible geneexpression system ; oxygen-dependent degradation ; genetherapy ; vascular endothelial growth factor
Abstract
STUDY DESIGN.: An in vitro neural hypoxia model and rat spinal cord injury (SCI) model were used to assess the regulation effect of a reporter or therapeutic gene expression by an oxygen-dependent degradation (ODD) domain in a hypoxia-inducible gene expression system with or without the erythropoietin (EPO) enhancer. OBJECTIVE.: To increase vascular endothelial growth factor (VEGF) gene expression in SCI lesions but avoid unwanted overexpression of VEGF in normal sites, we developed a hypoxia-inducible gene expression system consisting of the EPO enhancer upstream of the SV promoter and an ODD domain C-terminally fused to VEGF. SUMMARY OF BACKGROUND DATA.: ODD domain plays a major role in the degradation of hypoxia-inducible factor 1alpha and has been used in a hypoxia-specific gene expression system as a post-translational regulatory factor. METHODS.: The hypoxia-inducible luciferase or VEGF plasmid was constructed using the EPO enhancer combined with or without the ODD domain. The constructed plasmid was transfected into mouse Neuro 2a (N2a) neuroblastoma cells by Lipofectamine 2000, followed by a 24-hour incubation in hypoxia or normoxia. For in vivo analysis, the naked plasmid DNA was directly injected into the injured rat spinal cord. The gene expression was evaluated by luciferase activity assay, enzyme-linked immunosorbent assay, reverse transcriptase-polymerase chain reaction, and immunofluorescence staining. RESULTS.: The EPO enhancer/ODD domain-combined hypoxia-inducible gene expression system clearly increased the expression of the reporter luciferase gene and therapeutic VEGF gene specifically under hypoxic conditions and SCI, and quickly downregulated protein expression to a very low level after reoxygenation. CONCLUSION.: These results strongly suggest the potential applicability of this EPO enhancer/ODD domain-based hypoxia-inducible gene expression system in the development of a safer and more effective VEGF gene therapy for SCI
Full Text
http://ovidsp.ovid.com/ovidweb.cgi?T=JS&CSC=Y&NEWS=N&PAGE=fulltext&AN=00007632-200912150-00018&LSLINK=80&D=ovft
DOI
10.1097/BRS.0b013e3181c4af80
Appears in Collections:
1. College of Medicine (의과대학) > Dept. of Neurosurgery (신경외과학교실) > 1. Journal Papers
Yonsei Authors
Kim, Keung Nyun(김긍년)
Yoon, Do Heum(윤도흠) ORCID logo https://orcid.org/0000-0003-1452-5724
Ha, Yoon(하윤)
URI
https://ir.ymlib.yonsei.ac.kr/handle/22282913/105507
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