Effects Of DNA Methylation Level Of XIST Gene In Donor Cells On The Development Of Cloned Pig Embryos

Somatic cell nuclear transfer technology has played a significant role in promoting the development of animal husbandry,human biological medicine and life sciences.However,the development of this technique is limited by the low developmental efficiency of cloned embryos.It is believed that aberrant reprogramming of donor nucleus is one of primary reasons for the low efficiency of cloned embryos.Epigenetic reprogramming abnormalities present in various epigenetic modifications,including DNA methylation,histone modification,X chromosome inactivation,and genome imprinting.It has been reported that X chromosome inactive specific transcript factor,namely XIST gene,is implicated in X chromosome inactivation.The birth rate of cloned embryos was elevated by knockout XIST gene in the cloned mice and the cloned pigs.Our previous studies found that the DNA methylation levels in differentially methylated region of XIST gene(XIST-DMR)in cloned embryos may have a certain correlation with the DNA methylation levels of XISTDMR in the donor cells.Therefore,the aim of this study is to screen out single cell clones characterized by different DNA methylation levels of XIST-DMR,to compare effect of DNA methylation levels of XIST-DMR in donors on the developmental efficiencies of cloned embryos and DNA methylation levels of XIST-DMR at blastocyst stage.Based on above findings,we hope to establish a new approach to improve porcine cloning efficiency by screening appropriate somatic cell as donors.Firstly,34 single cell clones from the same porcine adult fibroblasts were obtained by single-cell inoculation.Secondly,single cell clones were distinguished by high and low DNA methylation levels on XIST-DMR which were measured through using the High Resolution Melting(HRM)method and the Bisulfite Genomic Sequence(BSP)method,and DNA methylation level of the single cell clones before freezing were found be the same with after resuscitation were proved by the BSP method.Finally,we compared the in vitro developmental efficiency of cloned embryos in which single cell clones with high and low DNA methylation levels on XIST-DMR as donors of nuclear transfer assays and effects of these two donor cells on DNA methylation levels on XIST-DMR of cloned blastocysts.Results revealed that the DNA methylation levels on XIST-DMR of donor cells had no significant effect on the in vitro development efficiency of the cloned embryos,and cloned blastocysts had low DNA methylation levels on XIST-DMR were identical to low DNA methylation levels on XIST-DMR of their donors.It is concluded that somatic cells with low DNA methylation levels on XIST-DMR is unable to improve in vitro developmental efficiency of cloned embryos,yet it can obtain cloned blastocysts with low DNA methylation levels of XIST-DMR.