| Summary: | 碩士 === 國立陽明大學 === 神經科學研究所 === 89 === The rat A2A adenosine receptor (A2A-R) is heavily enriched in the striatum and is also expressed at relatively low levels in other areas of the brain. Gene of A2A-R contains two independent promoters (P1 and P2). Transcripts produced from these two promoters differ in the length of their 5'-untranslated regions (5'-UTR), but encode the same protein. To further characterize the transcriptional regulation of the A2A-R gene in vivo, a fusion gene consisting the 4.8 kb 5'-flanking region / full length 5'UTR of the A2A-R gene and the coding region of the N-lacZ (n-β-galactosidase) was created to produce mice harboring the indicated fusion gene. Enzymatic analysis and histochemistry determinations reveal that the transgene (A2A-R promoter /β-galactosidase) was expressed mainly in the brain, indicating that the 4.8 kb 5'-flanking region / 5'UTR of the A2A-R gene might contain important elements for its expression in the brain. Furthermore, using the immunohistochemical double staining technique, I found that the transgene colocalized with the endogenous mouse A2A-R in various regions of the brain. Some of the transgene-positive cells are neurons and some are glial cells. Colocalization analysis of the transgene and various markers in the striatum shows that this transgene is located in both cholinergic and striatopallidal neurons (GABAergic neurons) as predicted. Nevertheless, the transgene-positive cells in the striatum where A2A-R is enriched only contributed to a small portion of the overall A2A-R-positive cells. To ascertain that the transgenic construct we designed contains the functional A2A-R promoters for its expression in the striatum, 5'-RACE experiments were carried out to evaluate the major functional A2A-R promoter in the striatum. To our surprise, results of 5'RACE suggest that additional exons and introns might exist for the rat A2A-R gene. Further analysis is required to clarify the complete gene structure of the rat A2A-R gene.
|
|---|
