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

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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.