Abstract
BackgroundThe complexity of the mouse mu opioid receptor (Oprm) gene was demonstrated by the identification of multiple alternatively spliced variants and promoters. Our previous studies have identified a novel promoter, exon 11 (E11) promoter, in the mouse Oprm gene. The E11 promoter is located ~10 kb upstream of the exon 1 (E1) promoter. The E11 promoter controls the expression of nine splice variants in the mouse Oprm gene. Distinguished from the TATA-less E1 promoter, the E11 promoter resembles a typical TATA-containing eukaryote class II promoter. The aim of this study is to further characterize the E11 and E1 promoters in vivo using a transgenic mouse model.ResultsWe constructed a ~20 kb transgenic construct in which a 3.7 kb E11 promoter region and an 8.9 kb E1 promoter region controlled expression of tau/LacZ and tau/GFP reporters, respectively. The construct was used to establish a transgenic mouse line. The expression of the reporter mRNAs, determined by a RT-PCR approach, in the transgenic mice during embryonic development displayed a temporal pattern similar to that of the endogenous promoters. X-gal staining for tau/LacZ reporter and GFP imaging for tau/GFP reporter showed that the transgenic E11 and E1 promoters were widely expressed in various regions of the central nervous system (CNS). The distribution of tau/GFP reporter in the CNS was similar to that of MOR-1-like immunoreactivity using an exon 4-specific antibody. However, differential expression of both promoters was observed in some CNS regions such as the hippocampus and substantia nigra, suggesting that the E11 and E1 promoters were regulated differently in these regions.ConclusionWe have generated a transgenic mouse line to study the E11 and E1 promoters in vivo using tau/LacZ and tau/GFP reporters. The reasonable relevance of the transgenic model was demonstrated by the temporal and spatial expression of the transgenes as compared to those of the endogenous transcripts. We believe that these transgenic mice will provide a useful model for further characterizing the E11 and E1 promoter in vivo under different physiological and pathological circumstances such as chronic opioid treatment and chronic pain models.
Highlights
The complexity of the mouse mu opioid receptor (Oprm) gene was demonstrated by the identification of multiple alternatively spliced variants and promoters
Generation of the transgenic mouse line In order to study both exon 11 (E11) and exon 1 (E1) promoters in mice, we made a ~20 kb transgenic construct in a pBR322 vector (Fig. 1)
Our previous studies indicated that the 3.7 kb region contained E11 promoter activity that was most evident in neuronal cells
Summary
The complexity of the mouse mu opioid receptor (Oprm) gene was demonstrated by the identification of multiple alternatively spliced variants and promoters. Our previous studies have identified a novel promoter, exon 11 (E11) promoter, in the mouse Oprm gene. The E11 promoter controls the expression of nine splice variants in the mouse Oprm gene. Alternative pre-mRNA splicing and multiple promoters of the Oprm gene have been hypothesized as molecular explanations of multiple mu opioid receptors. In addition to the rat MOR1B and human MOR-1A reported earlier[10,11], we have identified 25 splice variants from the mouse Oprm gene [12,13,14,15,16], which are derived from various combinations of sixteen exons that span over 250 kb. The functional significance of these splice variants has been suggested by differences in their region-specific and cell-specific expressions, agonistinduced G protein coupling and receptor internalization[12,14,17,19,20,21,22,23,24]
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