Production of knockout mice for serine racemase and analysis of the function of D-serine in central nerve system

大量的D-丝氨酸存在于人类和高等动物的大脑中。D-丝氨酸被认为与谷氨酸受体的N-甲基-D-天冬氨酸亚型结合并刺激神经传递。D-丝氨酸被认为是通过丝氨酸消旋酶从L-丝氨酸转化而来。然而，丝氨酸消旋酶和D-丝氨酸的功能还不清楚。因此，我们计划制备丝氨酸消旋酶基因敲除小鼠，以研究丝氨酸消旋酶缺陷的后果。为了制备丝氨酸消旋酶基因敲除小鼠，我们从小鼠基因组文库中筛选携带丝氨酸消旋酶基因的克隆，并分离出三个克隆。切下该基因的上游和下游区域，并将其插入含有新霉素抗性基因的基因靶向载体中。将构建的靶向载体线性化并转染到小鼠胚胎干（ES）细胞中。挑取新霉素抗性克隆并检查与丝氨酸蛋白酶的同源重组。 ...更多信息 消旋酶基因检查所选ES克隆是否保留了正常核型。通过显微操作，将来自三个重组ES克隆的细胞独立地接种到来自C57 BL/6小鼠的囊胚中。将囊胚接种到雌性ICR小鼠的子宫中，并出生三只嵌合体雄性小鼠。这些嵌合体小鼠与雌性C57 BL/6小鼠交配产生29只F_1杂合子小鼠。F_1代小鼠经兄妹交配产生65只F_2代小鼠，其中丝氨酸消旋酶缺陷型纯合子15只，杂合子35只，野生型纯合子15只。我们将观察这些小鼠的发育，并检测器官和血清中D-丝氨酸的水平。丝氨酸消旋酶的表达将通过实时PCR和蛋白质印迹法测定。此外，我们还将通过连续回交的方法，建立一个具有C57 BL/6遗传背景的丝氨酸消旋酶基因敲除小鼠品系。利用这些小鼠，我们试图阐明丝氨酸消旋酶和D-丝氨酸在中枢神经系统中的功能。少

A large amount of D-serine is present in the brains of human and higher animals. D-Serine is considered to bind to N-methy-D-aspartate subtype of glutamate receptors and to stimulate neurotransmission. D-Serine is considered to be converted from L-serine by serine racemase. However, the functions of serine racemase and D-serine are not well understood. Therefore, we have planed to produce serine racemase-knockout mice for the examination of the consequence of the serine racemase deficiency.In order to produce serine racemase-knockout mice, we screened mouse-genomic library for clones carrying the serine racemase gene and isolated three clones. Upstream and downstream regions of the gene were excised and inserted into the gene-targeting vector containing the neomycin-resistant gene. The constructed targeting vectors were linearized and transfected into mouse embryonic stem (ES) cells. Neomycin-resistant clones were picked up and examined for the homologous recombination with the serine … More racemase gene. The selected ES clones were checked that they retained the normal karyotype. The cells from the three recombinant ES clones were independently inoculated into blastulae from C57BL/6 mice by micromanipulation. The blastulae were inoculated into the uteri of female ICR mice and three chimeric male mice were born. Mating between these chimeric mice and female C57BL/6 mice produced 29 F_1 heterozygotic mice. Brother-sister mating of the F_1 heterozygotic mice produced 65 F_2 mice consisting of 15 serine racemase-deficient homozygotes, 35 heterozygotes and 15 wild-type homozygotes. We are going to observe the development of these mice and examine the level of D-serine in the organs and the serum. Serine racemase expression is going to be determined by real-time PCR and western blotting. In addition, we are going to establish a serine racemase-knockout strain with the genetic background of the C57BL/6 by continuous backcrossing of serine racemase-deficient mice with C57BL/6 mice. Using these mice we try to elucidate the function of serine racemase and D-serine in the central nerve system. Less