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

人类和高等动物的大脑中存在大量的D-丝氨酸。D-丝氨酸被认为与N-甲基-D-天冬氨酸受体亚型结合，并刺激神经传递。D-丝氨酸被认为是由L-丝氨酸通过丝氨酸消旋酶转化而来的。然而，丝氨酸消旋酶和D-丝氨酸的功能还不是很清楚。因此，我们计划建立丝氨酸消旋酶基因敲除小鼠，用于检测丝氨酸消旋酶缺陷的后果。为了产生丝氨酸消旋酶基因敲除小鼠，我们筛选了携带丝氨酸消旋酶基因的克隆，并分离了三个克隆。该基因的上游和下游区域被切除，并插入到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，并将其导入小鼠胚胎干细胞。挑选新霉素抗性克隆并检测其与丝氨酸…的同源重组更多的消旋酶基因。对筛选出的ES克隆进行了核型检测，证明它们保持了正常的核型。用显微操作法将3个重组ES克隆的细胞分别接种到C57BL/6小鼠的囊胚中。将囊胚接种到雌性ICR小鼠的子宫内，生下3只嵌合雄性小鼠。这些嵌合小鼠与雌性C57BL/6小鼠交配产生29只F_1杂合子小鼠。F_1代杂合小鼠兄妹交配产生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情况，并检测器官和血清中D-丝氨酸的水平。丝氨酸外消旋酶的表达将通过实时荧光聚合酶链式反应和蛋白质印迹来确定。此外，我们还打算通过丝氨酸消旋酶缺陷小鼠与C57BL/6小鼠的连续回交，建立一个具有C57BL/6遗传背景的丝氨酸消旋酶基因敲除株。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。较少

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