Cloned Embryos Mitochondria. Somatic Cell Nuclear Transfer Source Quantitative Analysis

Because human embryonic stem cells can grow infinitely in vitro and have the potential to differentiate into any cell types,they become a potential cell source for repairing human cells,organs and tissues.When used for transplantation,natural or IVF derived human embryonic stem cells will be rejected by patient's immune system because of immune incompatibility.Inhibitor of immunoreactions used for transplantation is toxic and very expensive. To solve the problem,our research team conducted a technical study of somatic cell nuclear transfer cloned embryos (Also known as therapeutic cloning).Somatic cell nuclear transfer can reprogram an adult cell into an original embryonic cell.This complex process is completed in enucleated oocyte and is an important scientific problem in therapeutic cloning research.Mitochondria of receptor cells and donor cells play an important role in cooperation of nucleo-cytoplasm,then research of receptor cell and donor cell mitochondrial dynamic changes in earlier developing period of SCNT-derived embryos will help for understanding its mechanism.Our study applied real-time PCR for detecting mitochondrial dynamic changes of receptor cell and donor cell in SCNT-derived embryos.The first part of our experiments is animal-animal SCNT research,the second part is animal-human SCNT research,and the last is human-human SCNT research.The following is the results.First,Using gene cloning technology,We cloned plasmid,containing DNA fragment of human,or rabbit,or mouse mitochondria and established quantitative real-time PCR methods to analyze mitochondria of human or rabbit using SYBR Green I dye fluorescence.Second,quantification showed that both human and rabbit mtDNAs coexisted in interspecies SCNT-derived embryos from l-cell to morula stages.These results suggest that maternal mtDNA replicates after the morula stage. Although donor cell mtDNA droppes to a very low level at the blastocyst stage,its mtDNA still exists.Third,copy numbers of both species mtDNA did not change significantly from l-cell to morula stages in mouse-rabbit cloned embryos.In blastocyst,however,the number of rabbit mtDNA increased sharply and the number of mouse mtDNA decreased.These results suggest that maternal mtDNA replicates after the morula stage.Fourth,in different developmental stages,quantitative analysis of mtDNAs revealed there is no significant difference between human 1VF-derived and human SCNT-derived embryos.