Abstract
The aims of this study were to evaluate the expression of enhanced green fluorescent protein (EGFP) driven by 6 different promoters, including cytomegalovirus IE enhancer and chicken beta-actin promoter (CAG), cytomegalovirus promoter (CMV), neuron-specific enolase promoter (NSE), myosin 7A promoter (Myo), elongation factor 1alpha promoter (EF-1alpha), and Rous sarcoma virus promoter (RSV), and assess the dose response of CAG promoter to transgene expression in the cochlea. Serotype 1 adeno-associated virus (AAV1) vectors with various constructs were transduced into the cochleae, and the level of EGFP expression was examined. We found the highest EGFP expression in the inner hair cells and other cochlear cells when CAG promoter was used. The CMV and NSE promoter drove the higher EGFP expression, but only a marginal activity was observed in EF-1alpha promoter driven constructs. RSV promoter failed to driven the EGFP expression. Myo promoter driven EGFP was exclusively expressed in the inner hair cells of the cochlea. When driven by CAG promoter, reporter gene expression was detected in inner hair cells at a dose as low as 3x10(7) genome copies, and continued to increase in a dose-dependent manner. Our data showed that individual promoter has different ability to drive reporter gene expression in the cochlear cells. Our results might provide important information with regard to the role of promoters in regulating transgene expression and for the proper design of vectors for gene expression and gene therapy.
Highlights
Gene transfer is a promising tool to study the physiology of the cochlea and cochlear cells
We systematically evaluated the promoters cytomegalovirus IE enhancer and chicken β-actin promoter (CAG), myosin 7A promoter (Myo), cytomegalovirus promoter (CMV), elongation factor 1 alpha promoter (EF-1 ), neuron-specific enolase promoter (NSE), and rous sarcoma virus promoter (RSV) for their abilities in gene transfers to the cochlear cells using in vivo assays
Robust enhanced green fluorescent protein (EGFP) expression with CAG promoter was shown in the HEK 293 cell, followed by CMV, NSE, EF-1 and RSV
Summary
Gene transfer is a promising tool to study the physiology of the cochlea and cochlear cells. The cytomegalovirus IE enhancer and chicken β-actin promoter (CAG) drives high level of transgene expression and is one of the most commonly used promoters for gene transfer (Xu et al, 2001). We have previously demonstrated that, with AAV1 vectors, the CAG promoter can drive transgene expression in the cochlea cells at a highly functional level (Liu et al, 2005). We systematically evaluated the promoters CAG, Myo, cytomegalovirus promoter (CMV), elongation factor 1 alpha promoter (EF-1 ), neuron-specific enolase promoter (NSE), and rous sarcoma virus promoter (RSV) for their abilities in gene transfers to the cochlear cells using in vivo assays. We examined the dose-response relationship for CAG promoter over a broad range in the cochlea
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