A novel adenoviral vector which mediates hypoxia-inducible gene expression selectively in neurons

Abstract

Selective gene expression in neurons is still a challenge. We have developed several expression vectors using a combination of neuron restrictive silencer elements (NRSEs), hypoxia responsive elements (HREs) and CMV minimal promoter (CMVmp). These elements were packaged into replication defective adenovirus to target gene expression selectively in neurons in a hypoxia-regulated manner. Neuronal selectivity and responsiveness to hypoxia of these novel constructs were determined empirically in both neural cell lines and primary cerebellar granule neurons (CGNs). The construct p5HRE-3NRSE exhibited not only the highest level of reporter gene expression in neuronal cells but also in an oxygen concentration-dependent manner when compared with all other constructs. As expected, this construct did not elicit reporter gene expression in non-neuronal cells including human HEK293A and HT29 cells, rat NRK cells, mouse 3T6 cells and 3T3 L1 cells. This construct was packaged into a replication defective adenoviral vector (Ad/5HRE-3NRSE) to determine neuron-selective and hypoxia-inducible gene expression in cultured mouse postmitotic primary CGNs and differentiated human NT2 neurons (NT2/Ns). Remarkably, in response to hypoxia, Ad/5HRE-3NRSE showed strong hypoxia-inducible gene expression selectively in neurons (12-fold induction in CGNs and 22-fold in NT2/Ns), but not in glial cells. Taken together, this vector with restricted gene expression to neurons under the regulation of hypoxia will be a useful tool for investigations of mechanisms of neuronal damage caused by ischemic insult.

CaMKII phosphorylates collapsin response mediator protein 2 and modulates axonal damage during glutamate excitotoxicity

“…In total, 10 lg DNA was used per transfection assay with the Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA). Briefly, cortical neurons plated in a 24-well plate were transfected at 7 days in vitro (DIV) as previously described (Huang et al 2005). The transfection efficiency is about 10% of the total number of cells in the dish.…”

Hypoxia-regulated neurotrophin-3 expression by multicopy hypoxia response elements reduces apoptosis in PC12 cells

“…Different copies of HRE can decrease the basal promoter activity under normoxic conditions. As shown in a previous study, the 5HRE-3NRSE regulator cassette decreased the basal CMVmp activity by approximately 50-97% in comparison with that of the CMV construct in cultured cells under normoxic conditions (19). Moreover, almost no humanized Renilla GFP (hrGFP) expression occurred in the PC12 cells infected by Ad-5HRE-hrGFP under normoxic conditions (20).…”

Neuroprotection of neurotrophin-3 against focal cerebral ischemia/reperfusion injury is regulated by hypoxia-responsive element in rats

“…First, HRE can decrease the basal promoter activity under normoxic conditions. It has been reported that a 5HRE-3NRSE regulator cassette decreased the basal cytomegalovirus minimal promoter (CMVmp) activity nearly by 50% and up to 97% in comparison with that of the CMVmp construct in cultured cells under normoxic conditions (Huang et al, 2005). Furthermore, almost no hrGFP expression occurred in PC12 cells infected by Ad-5HRE-hrGFP under normoxic conditions (Hu et al, 2009).…”

“…To assess the potential non-specific effects of the vector, reporter genes are commonly used, such as green fluorescent protein (GFP), b-Galactosidase (LacZ) and luciferase (Huang et al, 2005;Shen et al, 2006;Shi et al, 2009). We used EGFP as a system as well as a control for non-specific effects.…”