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

• 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
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06454182/
• 关键词: Catecholaminergic Neurons; Tyrosine Hydroxylase; Dopamine beta-Hydroxylase; Phenylethanolamine N-Methyltransferase; Adrenergic Receptors; Immunotoxin-Mediated Cell Targeting; Transgenic Mouse; Gene Targeting

(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）在DBH启动子控制下表达PNMT的肾上腺素亚型亚型 - 转基因小鼠的遗传改变和调节的表达被生成以切换儿茶酚胺特异性。在由转基因小鼠交感神经元支配的靶向组织中，beta2-ar结合位点的数量得到了显着改善，并且在组织中以不同的方式调节了beta1-ar结合位点。我们的数据表明，转基因小鼠中儿茶酚胺特异性的改变会导致目标时间中β-AR亚型的调节表达。 （2）多巴胺β-羟化酶的高水平表达和儿茶酚胺代谢 - 转基因小鼠的调节是用人类DBH Minigene构建体的多个副本生成的，以实现DBH的过表达。转化产物被正确处理至糖基化的糖基化的糖基化的聚二肽。人DBH在转基因小鼠中的表达导致酶的显着增加……在各种含有儿茶酚胺的组织中的活动更多的活性，但是通常维持去甲肾上腺素和肾上腺素的稳态水平，通常是在没有加速核甲胺胺的加速度的情况下维护的，而没有加速核胺的转移速率，这表明了某些固定率的固定速率，以提高固定剂的固定速率。 （3）在基因靶向基因产生的基因所产生的Th基因的酪氨酸羟化酶突变小鼠中的儿茶酚胺消耗和围产期致死性在胚胎发育的后期或出生后不久显示出致死性。该突变导致严重部署三种儿茶酚胺，但不会影响通常产生儿茶酚胺的细胞的总形态发展。对生存突变体的心电图分析表明心动过缓，表明纯合小鼠中心脏funciton的改变。另外，将人类TH转基因转移到纯合小鼠中，校正了突变表型，表明通过人酶的表达恢复了TH活性。我们的结果表明，在晚期胎儿发育和出生后，这对于动物的生存至关重要。 （4）免疫毒素介导的细胞靶向对特定神经元类型的条件破坏 - 我们已经开发了一种新型的转基因方法，可在大脑中消融具有免疫毒素的细胞毒性活性在大脑中诱导的选择性神经元。创建了转基因小鼠，该小鼠在DBH启动子的控制下表达人IL-2ralpha。对动物进行了I.C.V.具有免疫毒素抗TAC（FV）-PE40，它有选择地杀死带有人IL-2ralpha的动物细胞。免疫毒素仅在转基因小鼠中引起特征行为异常，伴随着含DBH的神经元的急剧丧失，DBH活性和去甲肾上腺素水平显着降低。这种方法提供了一种通用技术，可以通过靶标的人类神经退行性疾病创建动物模型

项目成果

K.Kobayashi: "Targeted disruption of the tyrosine hydroxylase locus results in severe catecholamine depletion and perinatal" J.Biol.Chem.(submitted).

K.Kobayashi：“酪氨酸羟化酶位点的靶向破坏会导致严重的儿茶酚胺消耗和围产期”J.Biol.Chem.（已提交）。

I.Nagatsu: "Expression of human tyrosine hydroxylase-chloramphenicol acetyltransferase fusion gene in the brains of transgenic mice by CAT immunohistochemistry" J.Neural Transm.96. 85-104 (1994)

I.Nagatsu：“通过CAT免疫组织化学在转基因小鼠大脑中表达人酪氨酸羟化酶-氯霉素乙酰转移酶融合基因”J.Neural Transm.96。

Kobayashi, K., Morita, S., Sawada, H., Mizuguchi, T., Yamada, K., Nagatsu, I., Watanabe, Y., Hata, T., Fujita, K., and Nagatsu, T.: "Targeted disruption of the tyrosine hydroxylase locus results in severe catecholamine depletion and perinatal lethality in

小林 K.、森田 S.、泽田 H.、水口 T.、山田 K.、长津 I.、渡边 Y.、畑 T.、藤田 K. 和长津 T.

K. Kobayashi: "Functional and high level expression of dopamine β-hydroxylase in transgenic mice" J. Biol. Chem.269. 29725-29731 (1994)

K. Kobayashi：“转基因小鼠中多巴胺 β-羟化酶的功能性和高水平表达”J. Biol. 2972​​5-29731 (1994)。

Kobayashi, K., Morita, S., Mizuguchi, T., Sawada, H., Yamada, K., Nagatsu, I., Fujita, K., and Nagatsu T.: "Functional and high level expression of human dopamine beta-hydroxylase in transgenic mice." J.Biol.Chem.269-47. 29725-29731 (1994)

Kobayashi, K.、Morita, S.、Mizuguchi, T.、Sawada, H.、Yamada, K.、Nagatsu, I.、Fujita, K. 和 Nagatsu T.：“人类多巴胺 β 的功能性和高水平表达