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）利用DIGE对肠、肝和血清中提取的蛋白质进行分析从GH转基因Amago的肠、肝和血清中提取蛋白质，利用DIGE对这些蛋白质进行蛋白质分析。通过双向凝胶电泳分离了约2000个蛋白质，并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，但消化酶和G蛋白的含量在转基因组中显著高于对照组。对肝组织的分析也表明转基因和对照之间存在巨大差异。转基因组中存在与寄生虫氨基酸序列相似的蛋白质，其含量是对照组的4倍左右。这些蛋白质仅检测到用于实验的所有转基因。（2）基因表达谱芯片分析方法我们用GH转基因小鼠和对照小鼠的肝组织进行消减杂交，并将消减后的DNA制成基因表达谱芯片。利用从未成熟和成熟GH转基因的F2和F3中提取的RNA进行微阵列分析。这些实验的结果表明，未成熟的幼鱼的自然免疫系统急剧下降。不过，这种倾向是在他们成熟的时候形成的。此外，在未成熟的转基因植株中也观察到一些与性别相关的基因表达，这些趋势在F2和F3中也有表现。（3）垂体基因的消减筛选以未成熟GH转基因鱼（F2）的垂体为材料，获得全长cDNA文库，并对未成熟GH转基因鱼进行消减杂交。筛选每1000个来自高表达和低表达的消减基因的克隆，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