Study on the role of histamine H1 receptors in the central nervous system using gene-targeted mouse

利用基因靶向小鼠研究组胺 H1 受体在中枢神经系统中的作用

基本信息

批准号：
13672289
负责人：
HORIO Shuhei
金额：
$ 0.64万
依托单位：
University of Tokushima
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13672289/
关键词：
histamine receptor desensitization phosphorylation down-regulation gene-targeted mouse gene targeting 遺伝子変異

项目摘要

To investigate the role of histamine H1 receptors in the central nervous system, we constructed a mutant human H1 receptor that does not undergo agonist-induced desensitization. This mutant was constructed by substituting possible phosphorylation sites of H1 receptor (Thr-140, Thr-142, Ser-396, Ser-398 and Thr-478 residues) to alanines. This mutation completely impaired agonist-induced down-regulation of the H1 receptor but it retained responsiveness of the receptor to histamine. Then we planed to construct genetargeted mice in which the wild type H1 receptor was replaced with this mutant H1 receptor. The level of the central H1 receptor will not be down-regulated upon continued stimulation with histamine. Thus the targeted mouse will show some abnormal behavior since its H1 receptor responsiveness to histamine will be exaggerated. To contruct the gene-targeted mice, we first obtained a BAC clone (C57/BL6 mouse) containing H1 receptor gene, and inserted a 13 kb fragment containing the gene into pBluescript vector. Then, we introduced mutations described above to the gene by PCR methods. Finally, we constructed a targeting vector by inserting a gene for positive selection (pGKNeopA) and a gene for negative selection (pMC1DTpA) to each appropriate site of the vector. The targeting vector was introduced to ES cells (TT2 cells) by electroporation, and approximately 400 colonies of ES cells were obtained. We are now identifying the clones whose H1 receptor gene is targeted using sutherm blot method. If we could obtain the gene-targeted ES cells, we will proceed to construct gene-targeted mice by injecting this ES cell to mouse blastocyte. This gene-targeted mouse will be very useful in clarifying the role of histamine H1 receptor in the central nervous system.

为了研究组胺H1受体在中枢神经系统中的作用，我们构建了一种突变的人H1受体，该受体不会经历激动剂诱导的脱敏。该突变体是通过将H1受体（THR-140，THR-142，SER-396，SER-398和THR-478残基）取代可能的磷酸化位点来构建的。该突变完全损害了激动剂引起的H1受体下调，但保留了受体对组胺的反应性。然后，我们计划构建遗传学小鼠，其中野生型H1受体被该突变的H1受体代替。通过组胺持续刺激，中枢H1受体的水平不会下调。因此，靶向小鼠将显示出一些异常行为，因为其H1受体对组胺的反应性将被夸大。为了构成靶向基因的小鼠，我们首先获得了一个含有H1受体基因的BAC克隆（C57/BL6小鼠），并将包含该基因的13 KB片段插入pbluescript载体中。然后，我们通过PCR方法将上述突变引入了基因。最后，我们通过插入一个基因进行阳性选择（PGKNEOPA）和一个用于阴性选择的基因（PMC1DTPA）来构建靶向载体。通过电穿孔将靶向载体引入ES细胞（TT2细胞），并获得了大约400个ES细胞的菌落。现在，我们正在使用Sutherm blot方法来识别其H1受体基因的克隆。如果我们可以获得靶向基因的ES细胞，我们将通过将该ES细胞注入小鼠胚泡来构建基因靶向小鼠。该靶向基因的小鼠在阐明组胺H1受体在中枢神经系统中的作用非常有用。