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

1)转基因猪的生产存在受精卵的制备、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，我们对卵母细胞成熟过程中发育的分子机制进行了研究。为了研究哺乳动物卵母细胞中MPF的激活是否需要GV，我们对猪未成熟卵母细胞进行了去核，并对其MPF活性和CCNB水平进行了研究。在去核的卵母细胞中，在成熟开始时检测到MPF的激活处于正常水平，但没有观察到重新激活以诱导第二次减数分裂。虽然蛋白质合成在质量和数量上都是正常的，但即使在去核的情况下，CCNB1也不能在去核的卵母细胞中充分积累。将GV材料注入去核卵母细胞，可逆转去核卵母细胞的缺陷。此外，对CCNB1降解的抑制表明CCNB1在去核卵母细胞中大量积累，表明CCNB1的合成活跃。丝裂原激活的蛋白激酶级联不受去核的影响。这些结果表明，GV不是第一次减数分裂激活MPF所必需的，而是第二次减数分裂所必需的，因为它促进了CCNB1的积累。2)本研究采用本实验室常见的分泌型白细胞蛋白酶抑制物(SLPI)作为靶向RNA干扰(RNAi)分子。我们研究了SLPI的RNAi对人癌细胞MCF-7和MDA-MB-231增殖的影响。我们还没有得到令人满意的结果的siRNA构建有效的RNAi。我们正试图为RNAi设计更有效的slpi siRNA结构

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