Immortalization of a fetal rat brain cell line that expresses corticotropin-releasing factor mRNA.

By introducing the SV40 T antigen under the control of promoter sequences from the gene encoding the rat corticotropin-releasing factor (CRF) into primary cultures from fetal rat brain, a stable cell line was established that expresses the SV40 T antigen and transcribes its endogenous CRF gene. The cell line exhibits neuronal properties, as demonstrated by positive immunostaining with antibodies against neuronal markers, and it provides a useful system for the detailed analysis of the transcriptional regulation of the neuropeptide gene at an early stage of development. Promoter activities of the CRF 5'-flanking region were tested in transient expression assays after transfection of the cell line with different fusion constructs with CRF promoter regions linked to the bacterial chloramphenicol acetyltransferase (CAT) gene. Basal activity of the promoter was determined by DNA sequences between positions -269 and -222 upstream of the transcriptional start site and showed weak induction upon treatment with forskolin.