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Basic study on development of transgenic farm animals for medical use by using RNA interference

利用RNA干扰开发医用转基因家畜的基础研究

基本信息

批准号：
16380197
负责人：
TOJO Hideaki
金额：
$ 9.98万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16380197/
关键词：
Gene knock down Gene manipulated farm animals Medical use-transgenic farm animals Galactose transferase RNA interference RNAi トランスジェニックブタ卵子 α-1,3-Gal EGFP トランスジェニックブタ

项目摘要

1)For production of transgenic pigs, there are many problems such as preparation of fertilized eggs, in vitro development of embryos and so on. As basic study on elevating the production efficiency of fertilized eggs, we have investigated molecular mechanism of development of oocytes during maturation. To examine whether the GV is required for MPF activation in mammalian oocytes, porcine immature oocytes were enucleated and their MPF activity and CCNB levels were investigated. The activation of MPF at the start of maturation was detected at normal levels in enucleated oocytes, whereas reactivation to induce the second meiosis was not observed. Although protein synthesis was found to be normal both qualitatively and quantitatively, even in the absence of the nucleus, CCNB1 did not sufficiently accumulate in the enucleated oocytes. The defects in the enucleated oocytes were reversed by the injection of GV material into the enucleated oocytes. Furthermore, the inhibition of CCNB1 degradation revealed drastic accumulation of CCNB1, indicating active synthesis of CCNB1 in enucleated oocytes. The mitogen activated protein kinase cascade remained unaffected by enucleation. These results indicate that GV is not required for the activation of MPF during the first meiosis, but that it is required for the second meiosis because of its promotion of CCNB1 accumulation. 2)In this study, SLPI(secretory leukocyte protease inhibitor) that has been well documented and is familiar protein in our laboratory was used for targeting RNA interference(RNAi). We have studied the effect of RNAi of SLPI on the proliferation of human cancer cells, MCF-7 and MDA-MB-231. We have not yet obtained satisfactory results of si RNA construct effective for RNAi. We are attempting to design more effective structure of siRNA of SLPI for RNAi

1)转基因猪的生产面临着受精卵的制备、胚胎的体外发育等诸多问题，作为提高受精卵生产效率的基础研究，本研究探讨了卵母细胞成熟过程中发育的分子机制。为了检查GV是否是哺乳动物卵母细胞中MPF激活所必需的，将猪未成熟卵母细胞去核并研究其MPF活性和CCNB水平。在去核卵母细胞中，在成熟开始时MPF的激活在正常水平下被检测到，而未观察到诱导第二次减数分裂的再激活。虽然蛋白质合成被认为是正常的定性和定量，即使在没有核，CCNB 1没有充分积累在去核卵母细胞。去核卵母细胞中的缺陷通过将GV材料注射到去核卵母细胞中来逆转。此外，CCNB 1降解的抑制揭示了CCNB 1的急剧积累，表明去核卵母细胞中CCNB 1的活性合成。有丝分裂原活化蛋白激酶级联反应不受去核的影响。这些结果表明，GV不是在第一次减数分裂过程中激活MPF所必需的，但它是第二次减数分裂所必需的，因为它促进了CCNB 1的积累。2）本研究利用本实验室已知的分泌性白细胞蛋白酶抑制剂SLPI（secretory leukocyte protease inhibitor）作为靶向RNA干扰（RNAi）的靶向蛋白。我们研究了SLPI的RNAi对人癌细胞MCF-7和MDA-MB-231增殖的影响。我们还没有获得对RNAi有效的siRNA构建体的满意结果。我们正在尝试设计更有效的SLPI siRNA结构，

项目成果

期刊论文数量（23）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Inhibitory function of whey acidic protein in the cell-cycle progression of mouse mammary epithelial cells (EpH4/K6 cells)

乳清酸性蛋白对小鼠乳腺上皮细胞（EpH4/K6细胞）细胞周期进程的抑制作用

DOI：
发表时间：
2004
期刊：
Journal of Reproduction and Development 50
影响因子：
0
作者：
Naito M;Minematsu T;Harumi T;Kuwana T;Naraolka H.;T.Unno et al.;Iwamori K;内藤充;S.Komori et al.;Sugiura K;H.Okamoto et al.;内藤充;Endo T;Naito M;M.Takayama-Ito et al.;Ito M;Naito M;Uekita T;Hiroyuki et al.;Ikeda K
通讯作者：
Ikeda K

Analysis of the promoter activity of the mutated whey acidic protein(WAP) gene

突变乳清酸性蛋白(WAP)基因启动子活性分析

DOI：
发表时间：
2006
期刊：
J Reprod Dev. 52
影响因子：
0
作者：
Naito;M.;Sano;A.;Kawashima;T.;Nakamichi;H.;Harumi;T.;Matsubara;Y.;Kuwana;T.;T.Sakamoto et al.;Ohtake Y;杉田昭栄;Nukumi Y
通讯作者：
Nukumi Y

Analysis of the promoter activity of the mutated whey acidic protein (WAP) gene

突变乳清酸性蛋白（WAP）基因启动子活性分析