Studies On Somatic Cell Nuclear Transfer Embryos Production And DNA Methylation

For the application of somatic cell nuclear transfer (SCNT), low cloning efficiency is a great barrier. In SCNT, the somatic nuclei must be remodeled from highly differentiated patterns to a totipotent embryonic pattern. DNA methylation is a major epigenetic modification and plays a crucial role in embryonic development and placentas'development. In this study, we detected the embryos'methylation status from 1cell to blastocyst of in vitro fertilization (IVF), bovine homospecies cloning and equus-bovine interspecies cloning. The study lays a foundation for studying the relation between DNA methylation and SCNT.1. The preparation of all stage of embryosâ‘ Acquired the reconstructed embryos by fusion bovine ear fibroblast or equus fetus fibroblast with enucleated bovine oocytes. Comparing the homospecies SCNT and interspecies SCNT, interspecies SCNT has a higher fusion rate than homospecies (82.36%vs75.58%), cleavage rate(85.32%vs88.41%)and blastocyst rate(4.38%vs35.69%).â‘¡Acquired the IVF embryos. Cleavage rate (86.25%) and blastocyst rate (46.87%).2. The Peg10 and H19 gene's methylation profiles in IVF and home-species cloning embryosâ‘ As the Peg10 gene, by comparing the homospecies SCNT with IVF, the result show that homospecies SCNT has a higher methylation level in 2-cell, morula and blastocyst (p<0.05) while lower level in 4-cell (p<0.01) and 8~16-cell(p<0.05). We presumed the interspecies SCNT had a process of earlier demethylation and de novo methylation. We presumed that all stages'higher methylation level, aberrant demethylation and de novo methylation may be an important reason of SCNT aberrant.â‘¡As the H19 gene, by comparing the homospecies SCNT with IVF, the result show that homospecies SCNT has a higher methylation level in 2-cell (p<0.05) and 4-cell (p<0.01) while lower methylation level in morula and blastocyst (p<0.01). We presumed homo-species SCNT mad the process of demethylation in morula or blastocyst and is an aberrant demethylation, so de novo methylation. The aberrant demethylation and intense wave of methylation level may cause the SCNT aberrant.3. The Peg10 and H19 gene's methylation profiles in inter-species cloning embryosâ‘ As the Peg10 gene, the interspecies SCNT's methylation level is similar to the homospecies SCNT except blastocyst stage, by comparing the IVF and homospecies SCNT's blastocyst methylation level, interspecies SCNT's has a lower level (p<0.01). We presumed that it undergo an aberrant demethylation and novo methylation in its development.â‘¡As the H19 gene, by comparing the IVF and homospecies SCNT, interspecies SCNT has a lower methylation level in 1-cell,2-cell and blastocyst (p<0.01), higher level in 8~16-cell (p<0.01). we presumed that the development undergo a earlier de novo methylation then an aberrant demethylation.