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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，我们构建了一个突变的人H1受体，不经历激动剂诱导的脱敏。该突变体通过将H1受体的可能磷酸化位点（Thr-140、Thr-142、Ser-396、Ser-398和Thr-478残基）替换为丙氨酸而构建。这种突变完全损害了激动剂诱导的H1受体下调，但它保留了受体对组胺的反应性。然后，我们计划构建基因靶向小鼠，其中野生型H1受体被替换为这种突变型H1受体。中枢H1受体的水平在组胺持续刺激后不会下调。因此，目标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。为了构建基因打靶小鼠，我们首先获得了含H1受体基因的BAC克隆（C57/BL 6小鼠），并将含该基因的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该基因。最后，我们通过将用于正选择的基因（pGKNeopA）和用于负选择的基因（pMC 1DTpA）插入载体的每个适当位点来构建靶向载体。通过电穿孔将靶向载体导入ES细胞（TT 2细胞），获得约400个ES细胞集落。我们现在正在确定克隆的H1受体基因的目标使用sutherm印迹方法。如果我们能够获得基因靶向的ES细胞，我们将通过将该ES细胞注射到小鼠囊胚中来构建基因靶向小鼠。该基因靶向小鼠的建立将有助于阐明组胺H1受体在中枢神经系统中的作用。