Role of Neurotransmitters and orphan receptors in neural cytoarchitecture of the brain..

神经递质和孤儿受体在大脑神经细胞结构中的作用。

基本信息

批准号：
11694322
负责人：
ICHINOSE Hiroshi
金额：
$ 2.56万
依托单位：
Fujita Health University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

Mice lacking Nurr1 failed to generate midbrain dopaminergic neurons, and die soon after birth. In order to dissect the molecular mechanism for Nurr1, we plan to knock-out Nurr1 and tyrosine hydroxylase, a rate-limiting enzyme in catecholamine biosynthesis, at the desired time at the specific organ using the inducible knockout system that is developed by Prof. Pierre Chambon and Dr. Daniel Metzger (our foreign collaborators).We screened the mouse genomic library constructed in lambda phage vector or bacterial artificial chromosome (BAC). We have isolated a Nurr1 gene, and constructed the floxed gene in which a crucial exon of the Nurr1 gene was flanked by loxP sites. We have obtained homologously recombinant ES clones.For the expression of the inducible recombinase Cre-ERT in dopaminergic neurons, a knock-in mouse line has been generated by introducing (via homologous recombination) Cre-ERT within the first exon of the tyrosine hydroxylase gene. Since tyrosine hydroxylase is a catecholamine-synthesizing enzyme, we expect that the mice specifically express Cre-ERT in tyrosine hydroxylase positive neurons. The mice were successfully created, and we are examining the expression of Cre-ERT by RT-PCR and Western blotting. We will cross the floxed mouse with mouse expressing Cre-ERT in catecholaminergic neurons, and examine the effect of inducible knock-out of the Nurr1 and tyrosine hydroxylase genes.

缺乏Nurr1的小鼠无法产生中脑多巴胺能神经元，并在出生后不久就死亡。 In order to dissect the molecular mechanism for Nurr1, we plan to knock-out Nurr1 and tyrosine hydroxylase, a rate-limiting enzyme in catecholamine biosynthesis, at the desired time at the specific organ using the inducible knockout system that is developed by Prof. Pierre Chambon and Dr. Daniel Metzger (our foreign collaborators).We screened the mouse genomic library constructed in lambda phage载体或细菌人造染色体（BAC）。我们已经分离了Nurr1基因，并构建了Floxed基因，其中Nurr1基因的关键外显子被LOXP位点侧面。我们已经获得了同源重组ES克隆。对于多巴胺能神经元中诱导型重组酶Cre-ERT的表达，通过在酪氨酸羟化酶基因的第一个外显子中引入（通过同源重组）CRE-ERT（通过同源重组）CRE-ERT产生了敲入小鼠系。由于酪氨酸羟化酶是一种儿茶酚胺合成酶，因此我们希望小鼠在酪氨酸羟化酶阳性神经元中特异性表达CRE-ERT。这些小鼠是成功创建的，我们正在研究RT-PCR和Western blotting的Cre-ert表达。我们将用在儿茶酚胺能神经元中表达CRE-ERT的小鼠穿越flox的小鼠，并检查Nurr1和酪氨酸羟化酶基因的诱导型敲除的影响。