Analysis of the murine 5-HT receptor gene promoter in vitro and in vivo

Abstract

The expression level of the 5-HT(1A) receptor gene (htr1a) in the central nervous system (CNS) is implicated in the aetiology and treatment of anxiety disorders and depression. Previous studies of the murine htr1a have revealed that its proximal promoter is GC rich and TATA-less. Several functional transcription factor binding sites, including MAZ and SP1 recognition sequences, have been identified. To further analyse the promoter of this receptor gene, additional upstream sequence information extending to -5.5 kb of the murine htr1a was generated and promoter fragments extending to -20 kb were analysed for activity in cell culture and transgenic animals. Promoter fragments greater than 4.5 kb in length were active in 5-HT(1A) receptor mRNA positive cells and inactive in 5-HT(1A) receptor mRNA negative cells. Smaller fragments were not able to confer this specificity. In agreement, using additive transgenesis to drive LacZ expression in vivo, CNS specific reporter gene expression was found with these longer constructs. Transgene expression in the 4.5- and 20-kb mouse lines resembled the endogenous htr1a expression pattern, whereas the 5.5-kb mouse lines surprisingly revealed strongly reduced expression. None of the three constructs was prone to confer ectopic expression, however, variation of expression between the transgenic lines was observed. Using colocalization studies we analysed the degree of concurrence of transgenic and endogenous htr1a expression brought about by these three different constructs. The highest degrees of colocalization were observed in mice harbouring the 20-kb construct, suggesting a large promoter fragment is required to faithfully direct transgene expression in a 5-HT(1A) receptor like pattern.