A novel chimeric promoter that is highly responsive to hypoxia and metals
To develop a potent hypoxia-inducible promoter, we evaluated the usefulness of chimeric combinations of the (Egr-1)-binding site (EBS) from the Egr-1 gene, the metal-response element (MRE) from the metallothionein gene, and the hypoxia-response element (HRE) from the phosphoglycerate kinase 1 gene....
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Published in | Gene therapy Vol. 13; no. 10; pp. 857 - 868 |
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Main Authors | , , , , , , , , , , |
Format | Journal Article |
Language | English |
Published |
Basingstoke
Nature Publishing Group
01.05.2006
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Subjects | |
Online Access | Get full text |
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Summary: | To develop a potent hypoxia-inducible promoter, we evaluated the usefulness of chimeric combinations of the (Egr-1)-binding site (EBS) from the Egr-1 gene, the metal-response element (MRE) from the metallothionein gene, and the hypoxia-response element (HRE) from the phosphoglycerate kinase 1 gene. In transient transfection assays, combining three copies of HRE (3 x HRE) with either EBS or MRE significantly increased hypoxia responsiveness. When a three-enhancer combination was tested, the EBS-MRE-3 x HRE (E-M-H) gave a hypoxia induction ratio of 69. The expression induced from E-M-H-pGL3 was 2.4-fold higher than that induced from H-pGL3 and even surpassed the expression from a human cytomegalovirus promoter-driven vector. The high inducibility of E-M-H was confirmed by validation studies in different cells and by expressing other cDNAs. Gel shift assays together with functional overexpression studies suggested that increased levels of hypoxia-inducible factor 1alpha, metal transcription factor-1 and Egr-1 may be associated with the high inducibility of the E-M-H chimeric promoter. E-M-H was also induced by hypoxia mimetics such as Co2+ and deferoxamine (DFX) and by hydrogen peroxide. Gene expression from the E-M-H was reversible as shown by the reduced expression of the transgene upon removal of inducers such as hypoxia and DFX. In vivo evaluation of the E-M-H in ischemic muscle revealed that erythropoietin secretion and luciferase and LacZ expression were significantly higher in the E-M-H group than in a control or H group. With its high induction capacity and versatile means of modulation, this novel chimeric promoter should find wide application in the treatment of ischemic diseases and cancer. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0969-7128 1476-5462 |
DOI: | 10.1038/sj.gt.3302728 |