Functional clarification of the bombesin-like peptide system.

1.缺乏NMB-R和GRP-R的小鼠的产生和分析：将雌性NMB-R+/-、GRP-R+/-小鼠和雄性NMB-R+/-小鼠杂交，获得雄性双基因敲除小鼠和雄性野生型小鼠。突变小鼠与野生型小鼠相比无明显差异。观察到的可能的变化是在GRP-R或NMB-R单一突变小鼠中已经注意到的点。高表达NMB-R、GRP-R和BRS-3的鸡胚的研制：构建了含有小鼠ReceptPr各亚型编码基因的逆转录病毒载体，并在其C端融合了EGFP基因。用这些载体制备的病毒颗粒感染鸡胚胎的脑区，导致了EGFP的强而广泛的表达。我们目前正在用组织学方法分析这些胚胎的表型。禽类蛙皮素系统分析：为了阐明禽类蛙皮素样肽系统，我们利用简并引物的RT-PCR方法，成功地从鸡脑中克隆了两个蛙皮素样多肽受体。我们将这些克隆命名为chGRP-R和chBRS-3.5，chGRP-R与其他物种的GRP-R具有很高的相似性，chBRS-3.5与BRS-3和Bb4相似。原位杂交结果显示chGRP-R在下丘脑和前脑的不同区域均有表达，chBRS-3.5在包括纹状体在内的前脑中广泛表达。这两种受体在小脑中均无表达。这些结果表明，蛙皮素样肽系统在鸟类中也存在，这表明该系统在鸟类中发挥着重要作用，就像哺乳动物一样。

1. Generation and analysis of mice lacking NMB-R and GRP-R : We obtained male double-knocked out mice as well as male wild-type mice by mating female NMB-R +/-, GRP-R +/- mice and male NMB-R +/- mice. The mutant mice did not show great difference as compared with wild-type mice. The possible alterations observed were the points already noticed in the GRP-R or NMB-R single mutant mice.2. Development of the chick embryos over-expressing NMB-R, GRP-R or BRS-3 : We constructed the retrovirus vectors containing cDNA coding for each subtype of mouse receptpr with EGFP cDNA fused at its C-terminal end. Infection of virus particles prepared using these vectors to the brain region of the chick embryos, resulted in the strong and wide-spread expression of EGFP. We are currently analyzing the phenotypes of these embryos by histological methods.3. Analysis of bombesin systems in birds : In order to elucidate the bombesin like peptides system in birds, we successfully cloned two bombesin-like peptide receptors from chick brain by using RT-PCR with degenerate primers. We named these clones as chGRP-R which showed high similarity with GRP-R of other species, and chBRS-3.5 which showed similarity with BRS-3 and BB4. In situ hybridization analysis showed that chGRP-R was expressed in the hypothalamus and in discrete regions of forebrain, and chBRS-3.5 was widely expressed in the forebrain including striatum. None of these two receptors was expressed in the cerebellum. These results demonstrated that the bombesin-like peptides system exists also in birds and this suggests that this system plays an important role in birds like in mammals.