| It is all along problems that efficiencies of somatic cell cloning was quite low in the fields ofporcine embryo-engineering, which subsequently limited the technique to produce usefulgenetic-modified pig. In order to resolve it, investigators need to know mechanisms of oocytematuration and early embryo development, so it's necessary to build systemic molecular anddevelopmental methods in the system. High-throughout gene screening has been applied in theexpression pool of oocye and early embryo, and many differently expressed genes and new geneshad been found, but knowledge about the specifically expressed genes are still limited. Among allmethods for gene function study, RNA interference (RNAi) is the strongest candidate because it isflexible, time-saving and available in oocyte and early embryo. RNAi was found in 1999 as amysterious phenomena in nametode and later in mammals, nevertheless it has surprisingly becomethe best tool in the research of gene function in the last short nine years, depending on itsunexampled technical predominance. Introducing RNAi technology into molecular anddevelopmental study in porcine embryo-engineering will provide theoretic basement forimprovement of in vitro porcine embryo production.The research primarily built an effective RNAi system to study the maternal expression pool ofporcine oocyte, by employing c-mos and Oct-4 gene as model gene, injecting of short interferingdsRNAs (siRNAs) as effector moleculars, as while as using Real time PCR and fluorescentlaser-confocal technology to detect the effect. In the mean time, the expression discipline andsystematic generating relation of c-mos and Oct-4 gene were studied respectively.Booth (2001) invented a new nuclear transfer method with zona-free bovine oocytes, which couldbe easily performed in rough experimental condition, and provided an alternative technique ofnuclear transfer. Cloned cattle and mice had been successfully born by applying zona-free oocytesystem. It is necessarry to study manipulating method of zona-free oocyte for improving theefficiency of the new cloning technique. Existence of zona pellucida imposed negative effects onthe immunochemistry dyeing as well as extracting protein and RNA of oocyte/early embryo. Thestudy investigated approaches to wipe off zona pellucida and voltages of electrical activation ofporcine oocyte, and provided method and basal data for the above research.Eleven conclusions were obtained:Among the three reagents for wiping off zona pellucida of porcine oocyte, proteinase K (PK) was evidently better than proteinase E (PE) in the viability and cyto-membrane intact situation aftertreatment, and was milder and more easily to manipulation than Acidic Tyrode's medium(A-TYR),so it was the best choice.In the experiment of parthenogenetic activation of ZPF oocyte, 110V waschosen to be the highest voltage, and 20V was the voltage grads. There were altogether fiveexperimental groups including 110V, 90V, 70V, 50V and 30V, along with a control group of 0V.Cleavage status situation was best in 50V, and blastocyst status was best in 90V.MOS and Oct-4 proteins were analyzed in the profile of molecular evolvement by drawingphylogentic tree with Neighbor-Joining method. And the molecular evolution relationships of thetwo protein were both accorded to the species evolvement discipline, and the two kinds oforthologs were also conserved respectively in functions. The homology of MOS existed in theeukaryote. And also have ortholog protein in two kinds of mouse sarcoma viruses; Oct-4homologies existed in invertebrate and vertebrate animal, but could not be found in avian species.Changes of c-mos mRNA level were detected by Real time PCR. mRNA of c-mos gene increasedduring the in vitro maturation (IVM) process, and decreased to 0.2034±0.150 times of the Oh of IVM6h after electric activation, and the decreasing trend continued till the embryo of two-cell stage. Theexpression and distributing changes of the protein were detected by immuno-fluorescence laserconfocal technology. At germinal vesicle (GV) stage, MOS had already expressed inside GV, andincreased before germinal vesicle breakdown (GVBD) and at the same tine began to dispersetowards cytoplasm, MOS level in the immatured oocytes was higher than matured oocytes at 44hrof culture, and there was little MOS in nucleus but a little in cytoplasm at 6hr after activation. Theinterfering sequences of siMOS803, siMOS1171 and siMOS 1311 could all knockdown the mRNAlevel of c-mos successfully to 0.084±0.03 and 0.114±0.06 and 0.204±0.06 times respectively to thecontrol at same stage. Although MOS is not depleted completely, but the quantity of it was obviousdecreased compared with control oocyte at the same stage and could induced chromosomes ofmatured oocytes to de-condensation without activation, and the less quantity of remaining MOS themore entirely of the de-condensation.The changes of Oct-4 mRNA level is detected by Real time PCR also. The results indicated thatOct-4 mRNA is continuously expressed through oocyte and early embryo and level of the mRNAwas increased before oocyte cleavage, decreased in cleavaged embryo and held on the low levelthereafter, and eventually increased rapidly in the blastula stage.The interfering sequences ofsiOct-4 466 and siOct-4 850 could both knockdown mRNA level of Oct-4, and were effectiveinterfering sequences, and the interfering effect of siOct-4 850 was better than siOct-4 466.Employing two effective interfering sequences of c-mos to detect the interfering effect of variousconcentration of the sequences, there were three groups for each interfering sequence: 0.2nM,0.2uM and 200uM, and the effects were detected after ld of interfering reaction. The results were:siMOS1311 could effectively interfere expression only in the highest concentration; andsiMOS1171 could effectively interfere expression both in 0.2nM and 200uM but failed in themiddle concentration of 0.2uM, which was not in the concentration-dependent manner. And the results also indicate that the effective concentration of a sequence have some relationship with theirtarget position and efficiency. Injection of siOct-4 850 to MⅡoocyte, the interfering efficiencycould reach to 60％in 1d, 90％or so in 2d, and there was no difference in interfering efficiencybetween 2d and 3d. RNAi reaction in porcine oocyte and embryo was time dependent.RNAi system established in the research could be applied in the study of maternal genefounction in time windows of maturation, arrest of MⅡstage and activation of oocyte as while asthe two cleavage before activation of zygote genome and so on.
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