Effects Of Rg108on The DNA Methyl Ation Level Of Buffalo Fibroblasts And Development Of Embryos Reconstructed With Them

Somatic cell nuclear transfer (SCNT) technology has been improved steadily in recent years, but the efficiency of nuclear transfer is still low, which limits the application of SCNT technology. It is well known that the incomplete dedifferentiation and reprogramming of donor nuclei in the oocytes-cytoplasm is the main factors leading to low efficiency in nuclear transfer. SCNT embryos often shows high levels of DNA methylation. In this study, the effects of RG108on the DNA methylation levels of buffalo fibroblasts and the development of SCNT embryos were investigated, so that to explore an effective way for improving nuclear transfer efficiency.Firstly, the effects of different concentration of RG108on cell growth, chromosome ploidy, the levels of DNA methylation and mRNA expression for DNMTs in buffalo fibroblasts were examined. The fibroblasts at passage7were treated with RG108(0,5,10,20,100μmol/L) for72h, respectively, then the state of cell growth and karyotype, as well as the level of DNA methylation, were detected and analyzed. The results showed that:(1) The growth curve of the buffalo fibroblasts treated with different concentration of RG108were consistent with the control group (0μmol/L), and there was not significantly different in the cell viability among of the groups of buffalo fibroblasts treated with different concentration of RG108(0,5,10,20,100μmol/L)(P>0.05).(2) Karyotype analysis found:it was no significant difference in the percentage of chromosome non-diploid (10.3%,12.3%,10.3%,12.1%,12.6%) when the donor cells treated with different concentration of RG108(0,5,10,20,100μmol/L).(3) The DNA methylation levels in the5-methylcytosine of buffalo fibroblasts were indirectly detected by immunofluorescence staining, and found that the levels of DNA methylation of the donor cells decreased corresponding to the increasing of RG108concentration, the20μmol/L group and the100μmol/L group was significant lower than that of the other three groups in the DNA methylation levels (P<0.05), but there was no significant difference between two groups (P>0.05).(4) The expression patterns of DNMTs in the donor cells were examined after RG108-treated by real time quantitative PCR (Q-PCR), and found that the mRNA expression levels of DNMT1in the groups of10,20,100μmol/L RG108was significantly decreased than that of0μmol/L group (P<0.05), and the20μmol/L group was the lowest than the others groups, but RG108had no an significant effect on the mRNA expression of DNMT3a (P>0.05).Meanwhile, the effects of RG108on the developmental ability of buffalo SCNT embryos derived from the donor fibroblasts treated with RG108were also examined. When the buffalo donor fibroblasts were treated with20μmol/L RG108for Oh,24h,48h,72h,96h, respectively, the cleavage rate of the reconstructed embryos among of each group was no significant diference (P>0.05), but the blastocyte development rate of reconstructed embryos from the donor cell treated with20μmol/L RG108for72h was significantly higher than that of the control group (0μmol/L, P<0.05), and the cell number of blastocysts from the group of the donor cell treated with20μmol/L RG108for72h was closer to the IVF group (P>0.05).These results suggested that:(1) The suitable concentration of RG108(≤100μmol/L) had no significant effect on cell growth characteristics, viability and karyotype.(2) Treating buffalo fibroblasts with20μmol/L RG108for72h could significantly reduce the level of DNA methylation and the mRNA expression of DNMT1, consequently improve the subsequent developmental ability of SCNT embryo and increase the cell numbers of blastocysts.