ESTABLISHMENT AND ANALYSIS OF SOMATOSTATIN RECEPTORS (SSTR2 AND SSTR5) KNOCKOUT MOUSE

生长抑素受体（SSTR2和SSTR5）敲除小鼠的建立与分析

• 批准号: 08671162
• 负责人: ABE Hiromi
• 金额: $ 1.6万
• 依托单位: KOBE UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08671162/
• 关键词: Somatostatin; Somatostatin receptors; Knockout mouse; GHRH; Growth Hormone; Pituitary; Hypothalamus; 2型ソマトスタチン受容体; 5型ソマトスタチン受容体; ジーンターゲッテング; 視床下部ペプチド

We subcloned genomic DNA fragment of somatostatin receptor, SSTR2 and SSTR5, with PCR using several sets of oligoprimers. 3.8Kb Fragment of SSTR2 and 4.2Kb fragment of SSTR 5 were obtained. Both fragment consist with small portion of 5'franking sequence and receptor coding regions. Neo-cassette was inserted into the coding region and the fragments were subcloned into the pMCtk vector with HSV-tK.Those vectors were transfected to the ES cells, then, clones were selected by Neo-resistance first followed by positive selection with acyclovir. After this step, 35 clones were selected and PCR with primers outside the homologously recombined site revealed that 3 out of 35 clones were successfully recombinated with trasfected DNA Fragment (two clones for SSTR2 and one for SSTR5). These 3 clones were injected to mouse embryo. We got heterozygous F0 judging from the fur patches, however, failed to get homozygous knockout mice in F1 generations.

我们利用PCR技术，用几组寡聚引物亚克隆了生长抑素受体SSTR 2和SSTR 5基因组DNA片段。获得了3.8Kb的SSTR 2片段和4.2Kb的SSTR 5片段。这两个片段都含有小部分5 '端的侧翼序列和受体编码区。将Neo-cassette插入编码区，将片段亚克隆到带有HSV-tK的pMCtk载体中，将这些载体转染ES细胞，首先通过Neo抗性筛选克隆，然后用无环鸟苷进行阳性选择。在此步骤之后，选择35个克隆，并且用同源重组位点外的引物进行PCR，显示35个克隆中的3个成功地与转染的DNA片段重组（两个克隆用于SSTR 2，一个用于SSTR 5）。将这3个克隆注射到小鼠胚胎中。F0代小鼠的毛斑为杂合子，F1代小鼠的毛斑为杂合子，F1代小鼠的毛斑为杂合子。

项目成果

Y Takahashi, H Abe, K chihara et al: "Biologically inactive growth hormone caused by an amino acid substitution" J Clin Invest. 100. 1159-1165 (1997)

Y Takahashi、H Abe、K chihara 等人：“氨基酸取代导致的生物无活性生长激素”J Clin Invest。

H Kaji, H Abe, K Chihara et al: "Novel compound heterozygous mutations of growth hormone(GH)receptor gene in a patient with GH insensitivity syndrome" J Clin Endocrinol Metab. 82. 3705-3709 (1997)

H Kaji、H Abe、K Chihara 等人：“GH 不敏感综合征患者中生长激素 (GH) 受体基因的新型复合杂合突变”J Clin Endocrinol Metab。

K Iida, H Abe, K Chihara et al: "Growth hormone (GH) insensitivity syndrome with high serum GH-binding protein levels caused by a heterozygous splice site mutation of the GH receptor gene producing a lack of intracellular domain" J Clin Endocrinol Metab.

K Iida、H Abe、K Chihara 等人：“生长激素 (GH) 不敏感综合征，血清 GH 结合蛋白水平升高，由 GH 受体基因的杂合剪接位点突变导致细胞内结构域缺乏”J Clin Endocrinol Metab

K Iida, H Abe K Chihara et al: "Growth hormone(GH)insensitivity syndrome with high serum GH-binding protein levels caused by a hoterozygous splice site mutation of the GH receptor gene producing a lack of intracellular domain" J Clin Endocrinol Metab. 83.

K Iida、H Abe K Chihara 等人：“生长激素 (GH) 不敏感综合征，血清 GH 结合蛋白水平升高，由 GH 受体基因的热合剪接位点突变导致细胞内结构域缺乏”，J Clin Endocrinol Metab。

Y Takahasshi, H Abe K Chihara et al: "Leptin induces tyrosine phosphorylation of cellular proteins including STAT-1 in human renal adenocarcinoma cells,ACHN" Biochem Biophys Res Commun. 228. 859-864 (1996)

Y Takahasshi、H Abe K Chihara 等人：“瘦素诱导细胞蛋白酪氨酸磷酸化，包括人肾腺癌细胞中的 STAT-1，ACHN”Biochem Biophys Res Commun。