| Summary: | 博士 === 國立陽明大學 === 神經科學研究所 === 90 === a2 adrenoceptors mediate the specific effect of epiniphrine and nor-epinephrine in various tissues via G protein-coupled signal transduction pathways. Converging evidence from biochemical, pharmacological and molecular cloning studies have confirmed the existence of at least 3 different subtype genes; Adra2a, (a2C10), Adra2b (a2C2) and Adra2c (a2C4). The existence of these receptor subtypes suggest that each may be endowed with different functions. In order to assess the differential expression pattern and physiological functions associated with one of the a2AR subtypes a2B in vivo; two approaches were employed; namely, the establishment of transgenic promoter-reporter mouse strains and the a2B knockout-lacZ knock-in mouse strains.
The temporal and spatial expression of a2B in the CNS was traced using transgenic (Tg) reporter mice. The transgene Adra2b-NN-lacZ is composed of a NcoI fragment (4.7 kb in length) immediately upstream from receptor coding region, and a reporter gene lacZ (encoding b-galactosidase). The selective expression of a2B in brain as indexed by b-galactosidase; under the direction of this promoter region, may be traced in situ using X-gal staining. The expression pattern of Adra2b-NN-lacZ in CNS of Tg mice during development was examined. The temporal course of examination was from gestation day 9.5 (E9.5) on to postnatal day 28 (P28). Significant X-gal staining was detected in the dorsal root ganglion (DRG), cranial nerves V and VII at E12.5. By E18.5, expression was noted in areas; cerebral cortex, anterior olfactory nucleus, hypothalamus, brain stem and cerebellar Purkinje cells etc., and persisted through postnatal development. Adra2b-NN directed reporter expression was detected in the hippocampal dentate gyrus first at P4. The temporal course of expression up to P28 in this area is in accordance with the developmental profiles of granule neurons of dentate gyrus. From P7 and on, transgene expression was detected in additional brain areas including septum and thalamus. The expression correlates well with the noradrenergic innervations as evidenced by co-localization using tyrosine hydroxylase (TH) or dopamine-b-hydroxyalse (DBH) immunocytochemsitry (ICC).
The second approach was aimed to establish mutant mouse strains deficient in a2B adrenoceptor subtype using a dual-purpose knock-out approach: Adra2b subtype gene was disrupted by a selection marker (Hprt) and a reporter gene (lacZ). The reporter gene was inserted such that its expression will be under the control of the respective regulatory sequences both up- and downstream of Adra2b gene. The mutant mice thus established will not only be used for functionality studies, but also serve as a much relevant model for tracing the temporal and spatial expression of a2B. Electroporation of Adra2b targeting construct into embryonic stem cell line (E14) was carried out. Targeted ES clones was selected by HAT (Hprt) and FIAU (HSV-tk) and validated by genomic Southern blot. Chimeric males obtained were subjected to breeding with C57BL/6J female mice for germline transmission check. Heterozygous Adra2b knockout mice were obtained and currently subjected to breeding for homozygous knockout mice. Spatial expression of a2B was traced in adult heterozygous Adra2b-KO/lacZ-KI mice by in situ X-gal staining. Forebrain, thalamus, hypothalamus and cerebellum are brain areas with significant a2B expression.
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