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

哺乳动物儿茶酚胺能神经系统功能的小鼠遗传学研究

基本信息

批准号：
06454182
负责人：
KOBAYASHI Kazuto
金额：
$ 4.93万
依托单位：
Fujita Health University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

(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

(1)儿茶酚胺特异性的遗传改变和肾上腺素能受体亚型的表达调节-产生在DBH启动子控制下表达PNMT的转基因小鼠，以改变转基因小鼠交感神经元支配的靶组织中β2的数量。 -AR结合位点显着减少，并且β1-AR结合位点在组织中以不同的方式受到调节。我们的数据表明转基因中儿茶酚胺特异性的改变。 (2)多巴胺β-羟化酶的高水平表达和儿茶酚胺代谢的调节-用多拷贝的人DBH小基因构建体产生转基因小鼠以实现DBH的过表达。转基因产物被正确加工成糖基化的成熟多肽的表达。转基因小鼠中的人类 DBH 导致各种含儿茶酚胺的酶活性显着增加(3)酪氨酸羟化酶突变小鼠的儿茶酚胺耗竭和围产期致死率——缺乏基因打靶产生的TH基因的小鼠在胚胎发育后期或不久后表现出致死率TH突变导致三种儿茶酚胺的严重消耗，但不影响正常儿茶酚胺的细胞的总体形态发育。对幸存突变体的心电图分析显示心动过缓，表明纯合子小鼠的心脏功能发生了改变。此外，将人类 TH 转基因转移到纯合小鼠中纠正了突变表型，显示通过人类酶的表达恢复了 TH 活性。 TH 对于动物在妊娠后期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条件性破坏特定神经元类型 - 我们开发了一种新的转基因方法，可以用产生了在 DBH 启动子控制下表达人 IL-2Rα 的转基因小鼠，并用免疫毒素抗 Tac 进行静脉注射治疗。 (Fv) -PE40，选择性杀死携带人 IL-2Rα 的动物细胞。免疫毒素仅在转基因小鼠中引起特征性行为异常，伴随着含有 DBH 的神经元的急剧损失以及 DBH 活性和去甲肾上腺素的显着降低。这种方法提供了一种通用技术，可以通过靶向Less来创建人类神经退行性疾病的动物模型。