Construction And Mitochondrial DNA Analysis Of Human-Bovine Interspecies Somatic Cell Cloned Embryos

The technique of interspecies somatic cell nuclear transfer, in which interspecies cloned embryos can be reconstructed by using domestic animals oocytes as nuclear recipient and human somatic cell as nuclear donor, can afford more opportunities in human embryonic stem cell and human organ transplantation by obtaining embryonic stem cell from interspecies cloned embryos which were cultured in vitro. But the application of this technique was limited by its extremely low efficiency which maybe can be attributed to donor nucleus not fully reprogrammed by xenogenic recipient cytoplasmy. Moreover, two types of mitochondria, which were derived respectively from nuclear donor and nuclear recipient, were observed in many interspecies cloned embryos and offspring. But the factors which affected mitochondrial heteroplasmy were still unclear. It should be considered that allogeneic mitochondria present in NT-ESC or NT-ESC derived cells could be recognized by the host immune system, leading to disrupted mitochondrial membrane potential that induces the apoptotic cell signaling pathway, thus leading to cell death.In present study, intra- and inerspecies cloned embryos were constructed using in vitro matured bovine oocytes as nuclear recipient and human- (HFFs) or bovine fetal fibroblasts (BFFs) as nuclear donor. The difference in developmental ability between intra- and interspecies cloned embryos were investigated by cultured them in vitro. Moreover, at various developmental stage (2-, 4-, 8-, morula and blastocyst) of interspecies cloned embryos, the percentage of two types of mtDNA and mtRNA was examined using PCR.A significant batch effect was found in construction of interspecies embryos. This showed that the cooperation between the enucleated oocytes and the donor cell is random. The cooperation is the most important factor in somatic cell nuclear transfer.The result of development in vitro showed that the rate of intra- blastocyst (10.1%) was higher than that of interspecies blastocyst (2.88%), and this difference was mainly due to lower developmental ability (p<0.05) of intra- than that of interspecies cloned embryos during 8- to morula.The results of analyzing on karyogene of interspecies embryos showed that the karyogene of interspecies embryos is from human donor cell.The results of examining two types of mitochondria in interspecies cloned embryos showed that before morula stage, the mtDNA from HFF can existed in interspecies cloned embryos; at all developmental stage, the mtDNA from bovine oocyte can existed in interspecies cloned embryos.