Analyse About Epigenetics Related Genes In Three Mouse Embryonic Stem Cell Lines

Somatic cells can be reprogrammed through transferring nuclear contents of somatic cell into oocytes, or by fusion with embryonic cells, or inserting several transcription factors (Oct4, Sox2, C-myc, Klf4 and Nanog) into somatic cell genome. This is a complex progress in which the most important is the epigenetic changs of the cell nucleus Here we discussed the recnt advance in the reprogramming of cell nucleolus, four different approach to generate embritic stem cells, the eptgenetic characteristic of stem cells in the morder epigenetic and the epigenetic changes reprogramming..Beginnine with the cultivation of ES cells, after a whole year hard work, mouse embryonic fibroblast(MEF ) in vitro cultivation platform was established , then based on this platform, The MEF was treated with mitomycin C,So the MEF was called feeder. Meanwhile, By injecting PMSG and HCG into mice for superovulation Then we washed the uterus to obtain blastocysts from the 3.5-day-pregnant mice, after that, those embryos were cultured on the feeder layers , cultivated these cells carefully in vitro to make sure them develop normally, then those cells notched. When inner cell mass developed larger,they obtained by mechanical method , As a result, established three mouse embryonic stem cell lines, which were examined and identified on molecular level.Since we have a relatively integrate embyronic stem cells culture system, Two ES cell lines, two nuclear transferring embryonic stem cells lines and two iPS cell lines was cultured in the laboratory . Then we extracted RNA from each cell line, and designed quantitive primers to analyse epigenetic genes relevant to epigenetic modification respectively, including Dnmt1, Dnmt3b, Dnmt3l and Aicda which were relevent to DNA Methylation; Ezh2, Kdm6b, Kdm6a relevant to H3K27me3; mllI, LSDI relevant to H3K4me; Kat2a relevant to Histone Ac.Using one ES cell line as control,we analysed differeces of those genes in these six cell lines, The result demonstrated that ntES2 haD a higher expression than other cell lines, especially Dnmt1, 5 times higher than other cells. As there were 25 passages of these cells cultured in vitro, so epigenetic changes of these cells may occur during in vitro passaging process, which had been reported previously, So extra indentification of this cell line was required. There were a little differences in Ezh2, Kdm6b, Kdm6a relevant to H3K27me3; mllI, LSDI relevant to H3K4me and Kat2a relevant to Histone Ac. In addition, there were no apparent differences of expression of Dnmt1 relevant to DNA Methylation in each cell line, however, 2 times lower expression of Dnmt3b occured in two iPS cell lines than other normal ES cell and nuclear trasferring stem cells. In contrast, approximately 4 times higher expression of Dnmt3L occured in two iPS cell lines than other normal ES cell and nuclear trasferring stem cells. In DNA methyltransferase aspect, iPS cell lines were different from normal ES cell and nuclear trasferring stem cells;, the extent of cell reprogramming had differences.