P-128: The Effect of DNA Methyl Transferase1 Inhibitor (RG108) on DNA Methylation Pattern of Cloned Mouse Embryos

Background: In somatic cell nuclear transfer (SCNT) method of cloning, transferred nucleus should be dedifferentiated from differentiated state to embryonic state. Molecular analysis showed that the reprogramming in the transferred nucleus was incomplete and cloned embryos have epigenetic abnormalities such as high DNA methylations levels. Since methylation in pre-implantation embryos has a critical role, hypermethylation or any other methylation disorders can alter the expression of genes related to the embryo development, that this changes in the later stages of fetal growth, appear to block development. Therefore, in this study for correcting epigenetic errors, the effect of RG108 as DNAmethyltransferase1 inhibitor on pre-implantation embryo development and methylation was assessed. And will discuss whether the RG108 can be reduced methylation in embryos obtained from cloning and increased potential of it. Materials and Methods: Two-cell stage embryos obtained from cloning, treatment with 5 and 10 μmol RG108 until morula stage and the pattern of DNA methylation was measured and analyzed quantitatively. Results: The percentage of development to the morula/ blastocyst stage in the group treated with 10 μmol RG108 (6.9 ± 3.3) were significantly lower in comparison to the non treatment group (35.2 ± 3.9). Also a significant difference was observed in developmental rate to the morula/blastosyst stage in the group treated with 5 μmol RG108 (17.9 ± 2.9) and non treatment group of cloned embryos (34.82 ± 3.99). But the difference between two treatment groups was not significant. Intensity of methylation between cloned embryos treated with 5 μmol RG108 (34.55 ± 3.19) and none treated (42.57 ± 5.99) showed significant differences, also the difference between normal fertilized (in vivo) embryos (36.53 ± 9.24) and those derived from cloning was significant while no significant difference showed between treated and in vivo embryos. Conclusion: Results of this study showed that treatment of mouse cloned embryos from 2-cell to morula stage with 5 μmol RG108, although it cannot increase to the level of development morula/blastosyst stage, but as one can possibly have a positive effect on correction of hyper methylation in the embryos obtained from SCNT.