Mouse genetic studies on the functions of the mammalian catecholaminergic nervous system

(1)儿茶酚胺特异性的基因改变和肾上腺素能受体亚型的调控表达——在DBH启动子的控制下，产生表达PNMT的转基因小鼠来切换儿茶酚胺特异性。在转基因小鼠交感神经元支配的靶组织中，β 2- ar结合位点数量显著减少，β 1- ar结合位点在组织间的调节方式不同。我们的数据表明，转基因小鼠儿茶酚胺特异性的改变导致靶组织中β - ar亚型的表达受到调节。(2)多巴胺-羟化酶的高水平表达和儿茶酚胺代谢的调控——利用人DBH基因的多个拷贝构建转基因小鼠，实现DBH的过表达。转基因产物被正确地加工成糖基化的成熟多肽。在转基因小鼠中表达人DBH后，该酶在各种含儿茶酚胺的组织中活性明显增加，但正常情况下，去甲肾上腺素和肾上腺素的水平维持在稳态，而儿茶酚胺的周转率没有加快，这表明尽管DBH蛋白的量增加，但去甲肾上腺素的合成速度保持不变。(3)酪氨酸羟化酶突变小鼠的儿茶酚胺缺失和围产期死亡率——基因靶向产生的缺乏TH基因的小鼠在胚胎发育后期或出生后不久就表现出致命性。TH突变导致三种儿茶酚胺的严重耗竭，但不影响正常产生儿茶酚胺的细胞的大体形态发育。存活突变体的心电图分析显示心动过缓，表明纯合子小鼠的心功能发生了改变。此外，将人TH转基因转移到纯合子小鼠中，纠正了突变表型，显示通过表达人TH酶恢复了TH活性。我们的研究结果表明，TH对动物在妊娠后期发育和出生后的生存至关重要。(4)通过免疫毒素介导的细胞靶向有条件地破坏特定类型的神经元-我们已经开发了一种新的转基因方法，通过免疫毒素的细胞毒性活性来消融大脑中诱导选择性的神经元。在DBH启动子的控制下，构建了表达人il - 2rα的转基因小鼠。这些动物用免疫毒素抗tac (Fv) -PE40进行体外治疗，这种免疫毒素选择性地杀死携带人类il - 2rα的动物细胞。免疫毒素仅在转基因小鼠中引起一种特征性的行为异常，这种异常伴随着含有DBH的神经元的急剧丧失，DBH活性和去甲肾上腺素水平的显著降低。这种方法提供了一种通过targetin Less创建人类神经退行性疾病动物模型的通用技术

(1) Genetic alteration of catecholamine specificity and regulated expression of adrenergic receptor subtypes-Transgenic mice expressing PNMT under the control of the DBH promoter were generated to switch catecholamine specificity. In the targeted tissues innervated by sympathetic neurons of transgenic mice, the number of beta2-AR binding sites was dramatically decreased, and beta1-AR binding sites were regulated in a different fashion among the tissues. Our data indicate that alteration of catecholamine specificity in transgenic mice leads to regulated expression of the beta-AR subtypes in the target tissues. (2) High level expression of dopamine beta-hydroxylase and regulation of catecholamine metabolism-Transgenic mice were generated with multiple copies of a human DBH minigene construct to achieve overexpression of DBH.The transgene products were correctly processed to a glycosylated mature polypeptide. Expression of human DBH in transgenic mice led to marked increase in the enzyme … More activity in various catecholamine-containing tissues, but the steady-state levels of noradrenaline and adrenaline were normally maintained without the acceleration of the catecholamine turnover rate, suggesting some regulatory mechanisms to preserve a constant rate of noradrenaline synthesis in spite of the increased amount of DBH protein. (3) Catecholamine depletion and perinatal lethality in tyrosine hydroxylase-mutant mice-Mice lacking the TH gene generated by gene targeting showed the lethality at a late stage of embryonic development or shortly after birth. The TH mutation resulted in severe depletion of three kinds of catecholamine, but did not affect gross morphological development of the cells that normally produce catecholamines. Analysis of electrocardiograms of surviving mutants showed bradycardia, suggesting an alteration of cardiac funcitons in the homozygous mice. In addition, tranfer of a human TH transgene into the homozygous mice corrected the mutant phenotype, showing recovery of TH activity by expression of the human enzyme. Our results represent that TH is essential for survival of the animals during the late gestational development and after birth. (4) Conditional disruption of specific neuronal types by immunotoxin-mediated cell targeting-We have developed a novel transgenic approach to ablate inducibly selective neurons in the brain with the cytotoxic activity of immunotoxins. Transgenic mice were created that express the human IL-2Ralpha under the control of the DBH promoter. The animals were treated i.c.v. with an immunotoxin anti-Tac (Fv) -PE40, which selectively kills animal cells bearing human IL-2Ralpha. The immunotoxin caused a characteristic behavioral abnormality only in transgenic mice, which was accompanied by a dramatic loss of DBH-containing neurons and a significant decrease in DBH activity and noradrenaline levels. This approach provides a general technique to create animal models of human neurodegenerative disorders by targetin Less