Research On Histone Deacetylase Inhibitors(TSA) For The Early Development Of Somatic Cloning Embryo

At present,the low efficiency of nuclear transfer(NT)and the development failure following NT are the capital bottleneck for the widespread application of the somatic NT technology.It was thought that the reprogramming disorder caused when somatic nuclei and oocyte cytoplasm interact following NT was the main reason.This experiment,repressed the histone deacetylation on different stage of early reconstructed embryo,using specific histone deacetylase inhibitor trychostatinA(TSA). By observing the effect of TSA treated on reconstructed embryo,we reveald the role of histone acetylation/deacetylation in reprogramming of somatic nuclei following nuclear transfer and provide important information for explaining the post-modificationf memory and fixed mechanism in reprogramming,and also try to find a new way that can improve nuclear transfer efficiency.Experiment 1:we study the effect of TSA concentration on development of reconstructed embryo.In according to related study,we desigh four groups of TSA concentration:0.5nM,5nM,50nM,500nM,respective ely.It was found that the blastocyst rate(up to 23.96%)with 50nM TSA treated was significantly higher than that of other four groups(11.94%,11.76%,14.29%,8.57%,respectively),and significant differences were existed(P＜0.05),which illustrated that 50nM TSA,on premise of minimum harm for reconstructed embryo,can utmostly inhibit histone deacetylase(HDAC),promote histone acetylation and thereby improve the development of reconstructed embryo.Experiment 2:we study the effect of histone acetylation/deacetylation treated on fuse stage on reconstructed embryo.This experiment aims to determine the histone acetylation/deacetylation treated on fuse stage on reprogramming and following development of reconstructed embryo.We desigh two groups:T3K6 group and K3K6 group.The result showed that there were no remarkable differences between two groups in 2-cell cleavage rate(81.82%vs83.18%)and blastocyst rate (18.19%vs21.45%,P＞0.05).It concluded that repressing histone acetylation on fuse stage can not improve the development of reconstructed embryo,i.e,there was no direct relationship between histone/deacetylation proceeded on fuse stage with nuclear reprogramming that influences the development of reconstructed embryo.Experiment 3:we investigate the effect of bistone acetylation/deacetylation treated on activation stage on reconstructed embryo.We desigh three groups:K3T6 group,K3K6 group and T3T6 group.It was found that the 2-cell cleavage rate(90.52%,91.04 %)and blastocyst rate(29.31%,32.54%)in K3T6 and T3T6 groups were significantly higher than K3K6 group(89.41%,21.65%),and significant differences were exisited(P＜0.05).But there was no remarkable differences between K3T6 group and T3T6 group in2-cell cleavage rate(91.04%vs90.52%)and blastocyst rate(29.31 %,32.54%,P＞0.05).It concluded that repressing histone acetylation on activation stage can improve the development of reconstructed embryo.Experiment 4:we investigate the effect of extension of activation time and that of TSA treatment on the development of reconstructed embryo.This experiment aims to observe whether different activation and TSA treatment time can affect the development of reconstructed embryo.We desigh foue groups:K2K6group,K2K9group,T2T6 group and T2T9 group,respectively.It was found that there were no remarkable differences between K2K6 group and K2K9 group in2-cell cleavage rate (83.51%vs86.89%)and blastocyst rate(18.56%vs19.67%,P＞0.05),and there were also no significant differences between T2T6 group and T2T9 group in 2-cell cleavage rate (87.38%vs88.34%)and blastocyst rate(31.06%vs334.19%,P＞0.05).It showed that extending activation and TSA treatment time can not improve the development of reconstructed embryo.Conclusion:(1)The best treated concentration of TSA is 50nM.(2)TSA,treated on fuse stage,did not contribute to the development of reconstructed embryo,showing that histone acetylation/deacetylation treated on fuse stage did not works for reconstructed embryo.(3)TSA,treated on activation stage,can significantly improve the development of reconstructed embryo,and hyperacetylation induced by TSA might improve reprogramming.(4)Extending activation and TSA treatment time can not improve the development of reconstructed embryo.