Analysis of aromatase/estrogen system in the age-related diseases by developmental engineering techniques

制备了完全丧失雌激素生物合成能力的芳香酶基因敲除(ARKO)小鼠，并对其大脑的性别分化进行了检测。结果表明，ARKO小鼠性动机、性行为、攻击性和亲子行为存在明显异常。Aromatae在小鼠大脑中的表达在围产期出现一个短暂的表达高峰，是大脑性别分化的标准，定位于特定脑区的神经细胞，即下丘脑的生殖中心、视前区和杏仁核。为了阐明脑芳香酶基因的神经元特异性、区域特异性和发育阶段特异性表达的调控机制，将芳香酶基因脑特异性启动子区域和β-半乳糖苷酶结构基因组成的杂交基因导入小鼠卵母细胞，获得转基因(TG)小鼠，并对转基因小鼠脑内β-半乳糖苷酶的表达进行了分析。…更确切地说，小鼠芳香酶基因外显子1上游至少6.4 kb的启动子区域对于脑芳香酶的神经元特异性、区域特异性和发育阶段特异性表达是必不可少的。人类芳香酶基因是通过多个外显子1和启动子的交替使用而受到组织特异性调控的。为了阐明组织特异性表达的调控机制，制备了携带报告基因(β-半乳糖苷酶)的人源化TG小鼠，该基因由人芳香酶基因的多个启动子控制。该报告基因在卵巢颗粒细胞、睾丸间质细胞、胎盘、胎肝、皮肤、骨动脉、脂肪组织中均有表达。报告基因在转基因小鼠体内的表达分布与芳香酶基因在人体内的表达分布完全相同。此外，交替外显子1a、1b、1c和1d在转基因小鼠组织中的组织选择性也与人类相同。这些结果表明，人源化的TG小鼠是分析人类芳香瘤基因组织特异性表达的有用的模型动物。较少

Aromatase knockout (ArKO) mouse which exhibits complete loss of capacity for estrogen biosynthesis was prepared and examined for sexual differentiation of the brain. The results indicated obvious abnormalities in the sexual motivation, sexual behaviors, aggressiveness, and parental behaviors of ArKO mice. Aromatae in the mouse brain shows a transient peak of expression at the perinatal period critaical for sexual differentiation of the brain, and is localized in nerve cells of the specific brain regions, namely of the reproductive center of hypothalamus, preoptic area, and amygdala. To elucidate the regulatory mechanism of neuron-specific, region-specific, and developmental stage-specific expression of brain aromatase, a hybrid gene cosisting of the brain-specific promoter region of aromatase gene and a beta-galactosidase structural gene was introduced into a mouse oocyte to create a transgenic (TG) mouse, and expression of beta-galactosidase in the brain of the TG mouse was analyzed. … More Conseqeuntly, at least 6.4 kb promoter region upstream from the brain-specific exon 1 of mouse aromatase gene is essential for neuron-specific, region-specific, and developmental stage-specific expression of brain aromatase.Human aromatase gene is tissue-specifically regulated by alternative use of multiple exons 1 and promoters. To elucidate the regulatory mechanism of tissue-specific expression, a humanized TG mouse carrying a reporter gene (beta-galactosidase) controlled by the multiple promoters of human aromatase gene was prepared. In this TG mouse the reporter gene was expressed in ovarian granulosa cells, testis Leydig cells, placenta, fetus liver, skin bone' artery, adipose tissue. The distribution of expression of the reporter gene in the TG mouse was exactly the same as that of the aromatase gene in human. Furthermore, the tissue-preference of alternative exons 1a, 1b, 1c, and 1d in the tissues of the TG mouse was also the same as that of human. These results indicate that this humanized TG mouse is a useful model animal for analysis of tissue-specific expression of human aromatae gene. Less