Construction of the neuron-specific expression vector carrying neural-restrictive silencer elements (NRSES) and its application to DNA transfection into primary cultured neurons

Abstract

Although the neuron-restrictive silencer element (NRSE/Regard) has been shown to function as a negative-acting DNA regulatory element to prevent the expression of neuron-specific genes in non-neuronal cells, little is known about its silencing effect on transcription in primary glial cells nor its effect on transcriptional activation in primary neurons. By DNA transfection in primary cultures of rat cortical neuronal or glial cells, we investigated the effect of NRSE on transcription mediated by the BDNF promoter I or c-fos promoter to which NRSE sequences derived from the SCG10 gene were linked. Transfection of plasmid DNAs to NIH3T3 fibroblasts resulted in a marked repressive effect of NRSE on BDNF promoter I- or c-fos promoter-mediated transcription. In primary neuronal cells, however, NRSE did not repress the basal promoter activities of BDNF and c-fos genes and allowed the transcriptional activation of these genes induced by membrane depolarization although NRSE slightly reduced the magnitude of BDNF promoter I activation. In contrast to neuronal cells, a marked repression of basal promoter activities of both genes was detected in primary glial culture and a two base pair-mutation of NRSE partially recovered the repression. These results indicate that NRSE negatively acts on its linked promoters in primary glial cells and does not interfere an activation of linked promoters in neuronal cells.