Isolation And Culture Of Bovine Embryonic Stem Cell-Like Cells And Effects Of Overexpression Of MIR-17-92Family On Their Target Genes Expression

The miR-17-92family can regulate and control the cell cycle and the apoptosis by down-regulating its target genes expression in posttranscriptional level. In this study, to understand the expression level of the miR-17-92family in bovine embryonic stem cell-like cells(bovine ES-like cells) and to investigate the effects of the overexpression of the miR-17-92family on bovine embryonic ES cell-like cells, the culture system of bovine ES cell-like cells was preliminarily founded. Then, the miR-17-92family gene was cloned, and a eukaryotic expression vector of the miR-17-92family was constructed. The expression level of the miR-17-92gene and the mRNA expression levels of E2F1and Bim in infected bovine ES cell-like cells were detected by the RT-PCR method. The study consisted of two parts:Firstly, the culture system of bovine ES cell-like cells was preliminarily established. The cell confluence degrees of feeder cells of four instruments were0%,40%,80%and100%, respectively. The primary colony formation rates of the groups of80%and100%were significantly different compared with the other two groups(P<0.05); but only the growth rate of diameter of primary clony in the80%group was the highest compared with the other three groups(P <0.05). Moreover, when the ES cell-like cells were respectively cultured in the feeder cells derived from bovine or buffalo fibrablasts or the mixture from the both of them, the primary colony formation rates were not significantly different among of three groups(P>0.05). Furthermore, the bovine ES cell-like cells from the passage method by instruments or by Accutase digestion could maintain plurypotency in12passages.Secondly, the expression levels of miR-17-92family members in bovine ES cell-like cells and the inhibitory effect of miR-17-92on target genes were studied. The cDNA of bovine ovary was used as templates, and the miR-17-92family gene which was1022bp was amplified by using PCR, and a fusion expression vector pLVX-IRES-ZsGreenl-miR-17-92was constructed, then it was used for lentivirus package and cell infection, and the overexpression levels of miR-17-92family members and the mRNA expression levels of target genes in bovine ES cell-like were detected by RT-PCR. The results showed that the the expression levels of miR-17-92gene family members were significantly up-regulated, but the mRNA expression levels of target genes E2F1and Bim were significantly down-regulated than those of control groups (P<0.05).In conclusions:the culture system of bovine ES cell-like cells is preliminarily established. The pLVX-IRES-ZsGreenl-miR-17-92vector is successfully infected into bovine ES cell-like cells by lentibirus, and it not only results in the miR-17-92gene overexpression but also down-regulates the expression levels of target genes E2F1in bovine ES cell-like cells.