Generation and characterization of an attenuated CMV promoter to drive low to moderate NRG expression in cultured neurons. A, Sequence of the attenuated CMV promoter. Bases shown in gray were removed. Locations of numerous cAMP response elements (CRE), a serum response element (SRE), and an AP-1 binding site that drive transcription, as well as the transcription initiation site (1), are indicated. B, Transcriptional activity was measured in HEK293 cells and in cultured hippocampal neurons cotransfected with reference plasmid pRL-TK (Promega) and either wild-type (WT) or attenuated (Att) CMV reporter constructs harboring firefly luciferase. Data are normalized to WT CMV and indicate the mean ± SEM of 3 (HEK293) or 4 (neurons) independent experiments run in triplicates (HEK 293) or duplicates (neurons). C, Subcellular distribution of NRG2 protein in hippocampal neurons transfected with constructs for GFP under the control of the Synapsin I promoter and for NRG2 under the control of the WT CMV promoter (left) or the attenuated CMV promoter (right). Signals were detected with NRG2 antibody mAB11. Immunoreactivity is widespread and blotchy in the neuron transfected with the WT CMV construct, whereas it is punctate and limited to the cell body and proximal neurites in the neuron transfected with the attenuated CMV construct. Scale bar, 10 μm.