DNA Methyltransferase In Buffalo-porcine Interspecies Somatic Cell Nuclear Transfer Reprogramming

Nuclear transfer(NT)is a crucial methods in studying cell reprogramming.In our research,we use the buffalo fibroblast as the donor cells,porcine oocytes as the receptor for interspecies somatic cell nuclear transfer(iSCNT).And plan to study DNA methyltransferase(Dnmt)'s function in iSCNT.We firstly analysis the karyotype of the cell lines from buffalo fetal.As a result,the cell lines we obtained had 48 chromosomes.This consistent with the karyotype of buffalo.We then used this cell lines for iSCNT.In order the assessed the potentiality development we used the parthenogenetic activation(PA)and normal SCNT as controls.There was significant difference between the iSCNT and SCNT groups in cleaved rate(77.19%vs 46.81%),and no significant difference between the iSCNT and PA groups(77.19%vs 66.29%).But in the blastocyst rate,the iSCNT groups had a very significant difference with the PA groups,and had significant difference with iSCNT group.It suggests that,iSCNT embryos genome activation(EGA)were failed.Secondly,we transfected cells with Green Fluorescent Protein(GFP)to iSCNT embryo,so that we can trace the dornor cell genome.We found that after iSCNT,the iSCNT embryo can fluoresce.This suggests that the dornor cell had been activated and remodeled.In addition,we optimized the embryo culture system of iSCNT embryos.And found that the PZM-3 were more appropriate than SOF-H as the culture medium(3.88%vs 0%).At last,we used the Dnmt inhibitor,5'-Azacytidine to inhibit Dnmt expression in donor cells to study the Dnmt function in iSCNT.As a result,we found that after Dnmt suppression in donor cells,the blastocyst rate was down regulation,but the difference was not significant.That suggested Dnmt in dornor cells play an subordinate role in reprogramming of buffalo-porcing iSCNT.