Effects Of Methyltransferase Inhibitor5-Aza-CdR Treatment On The In Vitro Development Of Debao Black Porcing Embryos Produced By Handmade Cloning

The indevelopment of cloned embryos is mainly due to uncompleted epigenetic reprogramming in the process of animal somatic cell cloning. For reducing DNA methylation level and improving reprogramming efficiency of cloned embryos, establishing and optimizating Guangxi Debao black pig handmade cloning (pig-HMC) technology system, in this study, we treated Guangxi Debao black pig ear fibroblasts and reconstructed embryos with DNA methyltransferase inhibitor5-Aza-2’-deoxycytidine (5-Aza-CdR), then investigated the proliferation and chromosome ploidy of donor cells, further more, statistically analysed embryonic development rate and methylation-related gene expression patterns of reconstructed embryos treated.1. Optimum5-Aza-CdR concentration for treatment of Debao pig ear fibroblasts was determined. In this study, the effect of Debao pig ear fibroblasts treated with different concentration (0μmol(L-1,0.25μmol·L-1,0.5μmol·L-1and1μmol·L-1) of5-Aza-CdR (DNA methyltransferase inhibitor) were investigated. The results showed that cells treated with0.25μmol·L-15-Aza-CdR have similar growth curve with that of the control group, and all treated cells have "S" shape of growth curve. With increasing of5-Aza-CdR concentrations, cells showed distortion, growth inhibition in varying degrees, and even death. Debao black pig fibroblasts treated with5-Aza-CdR from0μmol·L-1to0.5μmol·L-1didn’t show significantly alteration on the chromosome ploidy. Relative expression levels analysis show that the treatment of5-Aza-CdR on Debao black pig ear fibroblasts down regulated the expression of Dnmtl, Tetl, Tet2and Tet3and the regulating of Dnmtl, Tetl and Tet3was in a dose-related quantity pattern.2. The effects of donor cells and reconstructed embryos treated with5-Aza-CdR on in vitro developmental potency of Debao black pig handmade cloned embryos were investigated. Results showed that the cleavage and morula rates of handmade cloned embryos produced with donor cell treated with5-Aza-CdR had no significant difference with that of the control group.’However, the blastocyst rates of the treatment groups of0.25μmol·L-1and0.5μmol·L-15-Aza-CdR were higher than that of other groups, meantime there was a significant increasing on the total blastocyst cell number of pig-HMC in0.5μmol·L-1treatment group. Reconstructed embryos treated with5-Aza-CdR which concentrations applied in this study all can enhance the cleavage, morula and blastocyst rates of handmade cloned embryos. There was significantly increase on the blastocyst rates of20nmol.L-15-Aza-CdR treatment group and also the total blastocyst cells number of10nmol.L-1and20nmol.L-15-Aza-CdR treatment groups. While pig-HMC reconstructed embryos treated with20 nmol.L-15-AzA-CdR at different treatment time points (24h,48,72h and96h), the cleavage and morula rate were increased, and blastocyst rates and total blastocyst cell number were significantly increased in72h treatment group. However, when we treated the donor cells and reconstructed embryos with optimum time and concentration of5-AzA-CdR above in the same time, the developmental rate of reconstructed embryos and total blastocyst cells number were not increased.3. The relationship between epigenetic modification gene expression patterns and embryonic development potential on reconstructed embryos treated with5-Aza-CdR was investigated. Expression of Dnmtl, Tet1, Tet2, Tet3, Oct4and Nanog of handmade cloned embryos treated with5-Aza-CdR were detected by QRT-PCR. The results showed that the expression of Tetl, Oct4and Nanog were up regulated in handmade cloned embryos when5-Aza-CdR just applied on reconstructed embryos at1-cell stage, while Dnmt1, Tet2and Tet3were down regulated. At2-cell stage of handmade cloned embryo, the expression of Dnmtl, Tet2, Tet3and Oct4were up regulated, while Tetl and Nanog were down regulated. All gene expression patterns in the blastocyst stage were as same as that of2-cell stage, except Tetl was up regulated and Oct4was down regulated. When the donor cells and reconstructed embryos were treated with optimum time and concentration of5-AzA-CdR above in the same time, the expression of Dnmt1, Tetl and Tet3were up regulated, while Tet2, Oct4and Nanog were down regulated in1-cell stage of handmade cloned embryo. Then all gene expressing were down regulated in2-cell stage. However, in blastcyst stage, all gene expression patterns were as same as that of2-cell stage, except the expressing of Tet2, Oct4and Nanog were up regulated.Conclusion, donor cells treated with0.25μmol·L-15-Aza-CdR for72h and reconstructed embryos treated with20nmol·L-15-Aza-CdR for72h respectively can decrease the methylation level of cloned embryos and enhance in vitro developmental potency of Debao black pig HMC embryos.