Studies of complicated systems of growth using GH transgenic Amago

使用 GH 转基因 Amago 研究复杂的生长系统

• 批准号: 14360104
• 负责人: MORI Tsukasa
• 金额: $ 9.41万
• 依托单位: Nihon University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14360104/
• 关键词: GH transgenic Amago; subtraction; microarray; DIGE; 肝臓、腸、脳下垂体; F2,F3; GHトランスジエニックアマゴ; Ghrelin遺伝子; GHトランスジェニックアマゴF1; ニジマスGHS-R遺伝子; Ghrelin遺伝子組み換えニジマス

(1)Proteins analysis extracted from intestine, liver and serum using DIGEProteins were extracted from intestine, liver and serum of GH transgenic Amago, and these proteins were performed proteins analysis using DIGE. About 2000 proteins were separated by two-dimensional gel electro-phoresisis, and some of major proteins were extracted from the gel and performed MS/MS-analysis. Most of these were unknown proteins, however, amount of digestive enzymes and G-proteins were significantly greater in transgenic intestine than that of control. Analysis of liver tissue also showed vast differences among transgenic and control. There were proteins having similar to parasites amino sequences in transgenic, and these were about four times grater than control. These proteins were detected only all transgenic used for the experiment.(2)Analysis gene expression by microarrayWe have performed subtractive hybridization using liver tissue from GH transgenic and control, and made microarray plates using these subtracted DNA. Analysis of microarray was performed using RNA extracted from both F2 and F3 of immature and mature GH transgenic. The results of these experiments showed drastic decrease of natural immune system in immature young fish. However, this tendency was getting installed when they were matured. Further, some sex related gene expression was observed in immature transgenic, and these tendency were also showed both F2 and F3.(3)Screening of pituitary genes by subtractionThe completed length of cDNA library was obtained using pituitary of immature GH transgenic fish (F2), and also subtractive hybridization was performed using immature GH transgenic. Each 1000 clones of subtracted genes from both higher expression and lower expression were screened, and these have been sequenced.

(1)用DIGE对转GH的Amago进行肠、肝和血清蛋白的提取，用DIGE对提取的蛋白进行分析。用二维凝胶电泳法分离了约2000种蛋白质，并从凝胶中提取了部分主要蛋白质进行了MS/MS分析。其中大部分为未知蛋白，但转基因肠道中消化酶和g蛋白的含量显著高于对照组。对肝组织的分析也显示出转基因和对照组之间的巨大差异。结果表明，转基因蛋白中存在与寄生虫相似的氨基酸序列，且数量是对照组的4倍左右。这些蛋白仅检测到所有用于实验的转基因蛋白。我们对GH转基因和对照的肝组织进行了减法杂交，并用这些减法DNA制作了微阵列板。利用从未成熟和成熟GH转基因植株F2和F3中提取的RNA进行芯片分析。这些实验结果表明，未成熟的幼鱼的自然免疫系统急剧下降。然而，当他们成熟的时候，这种倾向就被安装了。此外，在未成熟转基因中也观察到一些性别相关基因的表达，这种倾向也表现为F2和F3。(3)垂体基因的减法筛选利用未成熟GH转基因鱼的垂体获得cDNA文库的完整长度（F2），并对未成熟GH转基因鱼进行减法杂交。从高表达和低表达的基因中筛选每1000个克隆，并对这些克隆进行测序。

项目成果

Localization of Ghrelin-Producing cells in the stomach of the rainbowtrout(Oncorhynchus mykiss)

虹鳟鱼 (Oncorhynchus mykiss) 胃中生长素释放肽产生细胞的定位

• 发表时间: 2004
• 期刊: Zoological Science 21
• 作者: Ichiro Sakata;Tsukasa Mori;Hiroyuki Kaiya;Mami Yamzki;Kenji Kagwa;Kinji Inoue;Takafumi Sakai.
• 通讯作者: Takafumi Sakai.

Localization of Ghrelin-Producing cells in the stomach of the rainbowtrout (Oncorhynchus mykiss).

虹鳟鱼 (Oncorhynchus mykiss) 胃中生长素释放肽生成细胞的定位。

• 发表时间: 2004
• 期刊: Zoological Science 21
• 作者: Ichiro Sakata;Tsukasa Mori;Hiroyuki Kaiya;Mami Yamzki;Kenji Kanagwa;Kinji Inoue;Takafumi Sakai
• 通讯作者: Takafumi Sakai