DNA Methylation Status Of Imprinting Genes In Lungs Of Somatic Cell Nuclear Transfer Bovines

Somatic cell nuclear transfer (SCNT) technology has been employed to produce animals of a wide variety of mammalian species,but the efficency of SCNT technique is very low,only 2-5%.The main problems in SCNT include high rates of pregnancy,perinatal and neonatal wastage,as well as abnormal offspring.It appears to be a great barrier to medicine industry,animal breeding and the propagation of rare animals.In SCNT,in order to support development,the differentiated state of donor nucleus requires epigenetic reprogramming to a totipotent,embryonic ground state.The incomplete or aberrant reprogramming of donor cell nuclei leading to aberrant expression or lack of expression of some developmentally important genes has been implicated as a primary reason for the low efficiency of SCNT.Genomic imprinting is an epigenetic phenomenon in which only one allele of a specific gene is expressed,depending on its parental origin.DNA methylation occurring on the 5 position of the pyrimidine ring of cytosines in the context of the dinucleotide sequence CpG is very important in the regulation of genomic imprinting.Most imprinted genes play important roles in embryogenesis and postnatal growth regulation.H19 gene is involved in the regulation of embryogenesis and fetal growth , Xist gene is a noncoding-RNA that plays a crucial role in X-chromosome inactivation,and Mash2 gene stimulates mononucleate trophoblast cell proliferation and inhibits binucleate cell formation.In order to make sure whether the DNA methylation reprogramming of imprinting genes is efficient in SCNT animals,we analyzed the DNA methylation status of three genes,H19,Xist and Mash2,in lungs of deceased SCNT bovines that died within 48 h of birth using bisulfite sequencing analysis.Our findings demonstrated that cloned bovines showed significantly lower DNA methylation of H19 than controls (P<0.05),and three tested CpGs sites (1,2,3) exhibited unmethylation in one cloned bovine (9C3).However,Xist showed similar DNA methylation levels between clones and controls,and both showed hypermethylation.The mean methylation levels of Mash2 in controls and clones were 20.04±9.99% and 5.55±5.36%,differences were considered to be statistically significant at P<0.05,and the percentage of overall mCpG in 9C5 was only 0.4%. These results showed that aberrant reprogramming of DNA methylation occurred in imprinting genes which correlated with development was ubiquitous.Therefore,aberrant DNA methylation may be a reason of the poor profile of SCNT and the organ abnormalities in SCNT bovines.