DNA Methylation And Hydroxylation Of Somatic Cell Cloned Cattle,Cloned Pig

With the development of cloning technology,low survival rate and phenotypic abnormality of surviving individuals have been paid more attention,Which are similar to the symptoms caused by aberrant of genomic methylation.5mC is an epigenetic modification associated with gene expression,but the function of 5hmC,which is involved in the process of DNA demethylation,is less known.In order to verify and further explore this issue,we choose 5mC and 5hmC,two closely associated epigenetic modifications,as subjects of this proposal.In this study,HPLC and LC-MS technology will be employed to examine the 5hmC?5mC content in different tissues of cloned and naturally reproduced animals.We think that this will deepen our knowledge and understanding of 5mC and 5hmC modifications in cloned cattle?cloned pig,and provide important clues to our understanding of the aberrant development of cloned animals.By using HPLC technology,we established a method for the detection of total DNA methylation(5mC)in tissues and cells.The methylation level of cloned cattle genome DNA was detected in different donor nuclei(FFB and FOV).The tissues we detect for cloned cattle include ear tissue,muscles,lungs,heart,spleen and liver.We established a method for the determination of total DNA(5hmC)content in tissues and cells by using LC-MS technology.In order to detect the methylation and methylation of cattle single locus in different donor nuclei,we measured the 5mC and 5hmC of imprinted genes H19 and Xist by using bisulfite(oxBS-seq)method.The results demonstrated that 5mC and 5hmC were both abnormal whether in live or dead cloned animals(cattle,pigs).The content of 5mC in survival cloned animals is higher than naturally reproduced animals but less than dead cloned animals and the content of 5hmC in survival cloned animals is higher than dead cloned animals but less than naturally reproduced animals.By comparing the imprinted gene H19 and Xist,we found the abnormal reprogramming of 5mC in H19 contributes significantly to the survival and death of cloned cattle.The content of H19 5mC in survival cloned cattle was generally lower than 50%,while the content of 5mC was about 70% in dead cloned cattle.Xist gene DNA 5mC was not significantly different no matter in live or dead cloned cattle.