| Epigenetic modification influences reprogramming and subsequent development of somatic cell nuclear transfer(SCNT)embryos. Such modification includes an increase in histone acetylation. Histone deacetylase inhibitors(HDACi), such as valproic acid (VPA) and scriptaid (SCR), have been known to maintain a high cellular level of histone acetylation. Hence, treatment of nuclear transfer embryos with HDACi may increase the efficiency of cloning. The present study attempted to examine the effects of VPA and SCR with regard to the potency of enhancement of in vitro and in vivo development of Wuzhishan miniature pig SCNT embryos.In CHAPTER1. The aim of the present study was to examine the effects of VPA, a HDACi on in vitro and in vivo development of Wuzhishan miniature pig SCNT embryos. Experiment1compared the in vitro developmental competence of nuclear transfer embryos treated with various concentrations of VPA for24h. Embryos treated with2mM VPA for24h reported a significantly increased rate of blastocyst formation compared with controls or4mM VPA and8mM VPA treated groups (21.5vs10.5,12.6and17.2%). Experiment2examined the in vitro developmental competence of nuclear transfer embryos treated with2mM VPA for different time periods following chemical activation. Embryos treated for24h reported higher rates of blastocyst formation than the controls or treated for4h and48h groups (20.7vs9.2,12.1and9.1%). In experiment3, an average of207(192to216) nuclear transfer embryos from the VPA-treated group were transferred to surrogate mothers, resulting in three pregnancies. Two of the surrogates delivered a total of11live piglets, the nine of11piglets in the VPA-treated group died in an unknown reason within1day to5days after birch. Untreated control embryos (average,205(179to225)) transferred to four surrogate mothers resulted in three pregnancies, two of which delivered a total of12live offspring, four of the12piglets in the VPA-untreated group died in an unknown reason within1day to3days, eight of12of the12piglets in the VPA-untreated group have survived over3or4months. The average birth weight of the two litters from the VPA-treated group was tended to be lower than that from the control groups (551.6g vs675.2g). These results suggest that VPA treatment increases the rate of blastocyst formation for SCNT embryos, both the VPA-treated and the untreated clones can develop to term in pigs, but the offspring from VPA-treated embryos survived to adulthood lower than the offspring from VPA-treated embryos(18.2vs66.9%; P<0.05).In CHAPTER2. The aim of the present study was to examine the effects of SCR, a HDACi, on the in vitro and in vivo development of Wuzhishan miniature pig SCNT embryos. Experiment1compared the in vitro developmental competence of nuclear transfer embryos treated with various concentrations of SCR for24h. Embryos treated with250nM SCR showed a significantly increased rate of blastocyst formation compared with controls or embryos treated with50nM or500nM SCR (20.0vs12.2,8.9, and11.9%). Experiment2examined the in vitro developmental competence of nuclear transfer embryos treated with250nM SCR for different time periods following chemical activation. Embryos treated for24h showed higher rates of blastocyst formation than controls or embryos treated for12h or48h (22.2vs11.5,10.0, and18.5%). Experiment3directly compared the effects of SCR and VPA treatment and examined the additive effect of SCR and VPA on nuclear transfer embryos following chemical activation. Embryos treated with250nM SCR or2mM VPA for24h showed a significantly increased rate of blastocyst formation compared with controls (19.9and18.3vs9.2%), but not increased with a combination of250nM SCR and2mM VPA for24h (18.9vs19.9,18.3%). In experiment4, nuclear transfer embryos treated with250nM SCR for24h and non-treated controls were transferred to surrogate mothers, resulting in the birth of healthy cloned pups from the controls but no births in the SCR-treated group. These results suggest that SCR treatment increases the rate of blastocyst formation in somatic cell nuclear transfer embryos and affects their subsequent growth. Also, co-treatment with VPA and SCR did not improve the blastocyst formation rate.In CHAPTER3. Genetically engineered pigs with cell markers such as fluorescent proteins are highly useful in lines of research that include the tracking of transplanted cells or tissues. It has been demonstrated that increased histone acetylation in SCNT embryos, by applying a histone deacetylase(HDAC) inhibitor such as VPA significantly enhances the developmental competence in our previous research. Herein, we report that the re constructed embryos treatment with2mM VPA for24h, and compared the development of SCNT embryos to the blastocyst stage when Wuzhishan miniature pig ear fibroblasts as donor cells, there have no significant differences were observed in the cleavage rates and blastocyste formation rates in non-trasfected or transfected ear fibroblasts (72.6,15.1and74.5,11.4%). we used the ear fibroblasts of miniature pigs transfected with EGFP as the donor cells, and transplanted reconstructed embryos (at the1to8cell stage) into the oviducts of three sexually mature Wuzhishan miniature pigs that were naturally estrus. One recipient successfully birthed two stillborn and two live piglets after116days of pregnancy. We investigated the presence of the foreign gene in the skin tissues of the piglets and tested13microsatellite polymorphisms in the recipient pigs, cloned offspring and donor cells. The results indicated that two cloned offspring expressed EGFP and all four cloned offspring had the same polymorphisms at all13microsatellites as the donor cell. However, the microsatellites varied between the cloned offspring and the recipient pigs, indicating the absence of a blood relationship between the two. The study therefore establishes a system to obtain transgenic Wuzhishan inbred miniature pigs by SCNT, thereby providing a basis to study the pathologic mechanisms of human disease and producing animal models for human genetic disorders. |
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