Studies of complicated systems of growth using GH transgenic Amago

(1)从肠道、肝脏和血清中提取DIGEProteins蛋白质，对转GH基因Amago的肠、肝和血清蛋白质进行DIGE分析。用双向凝胶电泳法分离了约2000个蛋白质，并从凝胶中提取了一些主要蛋白质并进行了MS/MS分析。其中大部分为未知蛋白质，但转基因肠道中消化酶和G-蛋白的含量显著高于对照。对肝组织的分析也显示出转基因和对照之间的巨大差异。转基因植株中含有与寄生虫相似的氨基酸序列，约为对照的4倍。基因表达的微阵列分析我们利用GH转基因和对照的肝组织进行了消减杂交，并用这些消减后的DNA制作了微阵列。用从未成熟和成熟GH转基因F2和F3中提取的RNA进行基因芯片分析。这些实验结果表明，未成熟幼鱼的自然免疫系统急剧下降。然而，这种倾向是在他们成熟时安装的。此外，在未成熟转基因鱼中还观察到了一些性别相关基因的表达，这些趋势在F2和F3中也表现出来。(3)用差减法筛选垂体基因。分别从高表达和低表达中筛选出1000个消减基因克隆，并对这些克隆进行了测序。

(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.