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

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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