Study On Production Procedure Of Cloned Piges And Transgenic Piges By Somatic Cell Nuclear Transfer

Somatic cell nuclear transfer (SCNT) technology has been widely used in animal agriculture and biomedical studies. The production of cloned pigs by SCNT includes several steps, The important steps affecting the cloning efficiency are the enucleation of the recipient oocyte and the epigenetic modification of reconstruct embryo, moreover, the comparison of transgenic and non-transgenic cell has also been a lot of controversy. This experiment set about three aspects, include the effect of Demecolcine(DEM)-assisted enucleation on maturation promoting factor (MPF) levels and Cyclin B1 distribution in porcine oocytes, the efferct of one histone deacetylase inhibitor on in vitro developmental competence of pig somatic cell nuclear transfer embryos, and the compare of difference between transgenic and non-transgenic clone pig, to further improve the development potential of somatic cell cloned embryo, optimize production outfit to somatic cell nuclear transfer and transgenic pigs operating procedure.Experiment 1 Demecolcine is a microtubule disrupting agent, in many species have been widely used in the enucleation of M Ⅱ oocytes. Nevertheless, there have been no reports regarding the effect of this method on distribution of MPF in porcine oocytes. Result:(1) The effects of the DEM treatment time on MPF levels were examined. In our study, oocytes were treat with DEM for 0.5,1,2, or 3 h. The optimal concentration of DEM to induce ooplasmic protrusions in porcine oocytes was 0.4 μg/ml. (2) Here, we assessed MPF levels and Cyclin B1 distribution in porcine oocytes after DEM treatment. Oocytes treated with 0.4 μg/ml DEM for 1 h exhibited a uniform distribution of MPF in the oocytes. Immunofluorescence microscopy showed that Cyclin Bl, the regulatory subunit of MPF, was extremely abundant around nuclear spindle, but rare in the cytoplasm of control oocytes. DEM treatment disrupted spindle microtubules and induced chromosome condensation, and reduced Cyclin B1 in the nuclear region. Cyclin B1 was uniformly distributed in DEM-treated oocytes, leading to increased MPF levels. The developmental potential in vivo was significant higher than control group. This result is the first report of the effect of DEM-assisted enucleation on Cyclin B1 distribution in porcine. MPF in mature oocytes is an important factor for supporting the development of the reconstructed embryo, and is crucial for the efficiency of SCNT. (3) Enucleation efficiency and development of NT embryos in porcine oocytes by DEM-assisted enucleation or mechanical enucleation. The MPF activity in oocytes enucleated by DEM-assisted enucleation were significant higher than those obtain by mechanical enucleation. The efficiency of enucleation of DEM-treated porcine oocytes was 98.3% and was significantly greater (P< 0.05) than the efficiency of oocyte enucleation by blind aspiration (76.1%). The developmental potential of NT embryos was examined using enucleated porcine oocytes as recipient cytoplasts. The proportions of NT oocytes receiving pig ear fibroblasts that developed to 2-4 cell and blastocyst stages were not significantly different between DEM-assisted enucleation and mechanical enucleation (16.2% vs.14.1%). The development potential in vivo of DEM-treated group was significantly greater than control group.Experiment 2 The low success rate of animal cloning by SCNT associates with epigenetic modification of reconstruct embryo, including the abnormal acetylation of histones. Altering epigenetic status by histone deacetylase inhibitors (HDACi) enhances the developmental potential of SCNT embryos. HDACi, such as CUDC-101, have been known to enhance the level of histone acetylation, but yet never used in SCNT embryos. In present study, for the first time, attempted to examine the effect of CUDC-101 after the pig somatic cell nuclear transfer embryos and determinate optimal concentration and duration. To selected optimal concentration, the pig SCNT embryos were treated with different concentrantions of CUDC-101 for 24 h, and pig SCNT embryos were treated with optimal concentration for different time. In addition, CUDC-101 treated pig SCNT embryos were transferred into the oviducts of surrogates on the day on which estrus began or 1 day after. Result:We found that treatment with 1/uM CUDC-101 for 24 h significantly improved the development of pig SCNT embryos. Compared with the control group, the blastocyst rate was higher (18.1% vs.9.9%,P<0.05).Experiment 3 Generation of transgenic pigs by somatic cell nuclear transfer has provided a new avenue to modify disease resistance or susceptibility in pigs. In this study, we evaluated the efficiency of producing cloned transgenic piglets by SCNT. Donor cells were isolated from a 2-year-old female Wuzhishan miniature piglet. Cells transfected with 3 different types of disease resistance gene (anti-viral interferon-y gene (IFN-y), anti-mouth disease virus single chain antibody gene (scFv) and swine IFN-y/scFv fusion gene(IS)), either transfected and non-transfected cells between passages 3 and 5 were used for cloning. Result:(1) We examined the developmental potential of porcine nuclear transfer embryos when IFN-y, scFv, IS and non-transfected cells were used as donor cells. There was no significant difference in the developmental rate of among 3 different types (IFN-y, scFv, IS) NT embryos to the 2-4 cell stage and non-transgenic donor cells (78.4%,79.5%,79.7% vs.80.6%, respectively, P<0.05). Furthermore, there was no significant difference in the developmental rate of NT embryos to the blastocyst stage and transgenic or non-transgenic donor cells (10.8%,12.0%,11.4% vs.16.2%, respectively, P<0.05) (2) while cloned embryos reconstructed with 4 different types of donor cells were introduced into 27 surrogate mothers. Among these mothers,19 were pregnant, as detected by ultrasonography on days 24-26 following embryo transfer.11 surrogate mothers gave birth to 63 live and 4 stillbirth naturally cloned piglets after 114-120 days of gestation.25 piglets were died of an unknown cause afer lweek. In final, we used 3 different types (IFN-y, scFv, IS) of transgenic cells as donor cells to reconstruct NT embryos by SCNT and give birth 49 cloned pigs, but only six PCR-positive founders were identified. We successed to product 6 transfer of IFN-y or scFv gene Wuzhishan inbred miniature pigs by somatic cell nuclear transfer, to provide a suitable approach to produce resistant transgenic pigs.