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

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，它不会经历激动剂诱导的脱敏。该突变体是通过将H1受体可能的磷酸化位点(Thr-140、Thr-142、Ser-396、Ser-398和Thr-478残基)替换为丙氨酸而构建的。这种突变完全削弱了激动剂诱导的H1受体的下调，但它保留了受体对组胺的反应性。然后我们计划构建基因定位的小鼠，在其中野生型H1受体被这个突变的H1受体取代。中枢H1受体的水平不会因组胺的持续刺激而下调。因此，目标小鼠将表现出一些异常行为，因为它的H1受体对组胺的反应性将被夸大。为了构建基因靶向的小鼠，我们首先获得了含有H1受体基因的BAC克隆(C57/BL6小鼠)，并将含有该基因的13kb片段插入到pBluescrip载体中。然后，我们通过聚合酶链式反应的方法将上述突变引入到该基因中。最后，将正选择基因(PGKNeopA)和负选择基因(PMC1DTpA)分别插入到载体的适当位置，构建了靶向载体。通过电穿孔将靶向载体导入ES细胞(TT2细胞)，获得了约400个ES细胞集落。我们现在正在用Sutherm印迹法鉴定其H1受体基因为靶点的克隆。如果我们能获得基因靶向的ES细胞，我们将通过将该ES细胞注射到小鼠的囊胚细胞中来构建基因靶向的小鼠。这种以基因为靶标的小鼠将非常有助于阐明组胺H1受体在中枢神经系统中的作用。

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.