| Embryonic stem cells (ESCs) have capability to self-renew and differentiate into cells of all three germ layers, distinguishing them from other stem cells. ESCs have been extensively applied in development research, tissue regeneration, and so on. RNA interference (RNAi) is one of the most important techniques to study gene functions. Conventional chemical methods for gene delivery were either low transfection efficiency or high toxicity when applied in ESCs; however, lentiviral vector can infect ESCs with high efficiency and low immunoreactions. We started this research by the establishment of a high efficiency lentiviral RNAi system to discover the function of the potential self renew genes of mouse ESCs (mESCs) such as DUSP5.Viral plasmid (pLKO.1), packaging plasmid (psPAX2), and envelop plasmid (pMD2.G) were co-thansfected into HEK293T cells with PEI. Viral with high infection efficiency can be harvested every 12h after 24h thansfection, for total 4-6 times. We valued the gene delivery ability of PEI, and improved the method of thansfection by PEI, such as the ratio of PEI to plasmid is 12:1 (w:w), PEI dose not exceed 25μg/ml, treatment time is 10-12h. We also constructed a new lentiviral vector which have EGFP report gene in pLKO.1 cloning vector. It could be more convenient to screen cloning by fluorescence microscopy as the puromycin resistance gene (puroR) was replaced by EGFP. Viral in the present study had successfully infected HEK293/293T, K562, A02, NIH3T3, MEF, mES D3 and mES Zju-J1 cells, and the infection cell lines have been establishedRNAi was tested by the interference of EGFP, both in HEK293T and mES cells. The interference efficiency of EGFP could be 87.4%, performing in EGFP HEK293T cells. We have selected one shRNA from 3 sequences designed for DUSP5, which got 59.3% interference efficiency in mES D3 cells. We also selected one shRNA from 2 sequences designed for DUSP1, which got 90.5% interference efficiency in mES D3 cells. The same was to AIRE, which got 74.3%.Nanog expression level in mES D3 cells was decreased, and phosphorylation level of ERK increased after DUSP5 interference. It was consistent with the dual-specificity ERK phosphatase activity of DUSP5, and suggested that the potential relative of DUSP5 to self renewal of ES cells. DUSP1 expression level was down-regulated after DUSP5 interference, and DUSP5 expression level was also reduced after DUSP1 interference, which told us that the two genes may be linked in mES cells.Nanog, Oct4 expression was decreased, after AIRE interference in mESCs. The interfered cells partly lost the capability of cloning formation. Growth rate was also lower, as comparing with normal mES D3 cells. Cell cycle testing show that the S phase greatly decreased and the G2/M phase increased. We conclude that AIRE would be an important factor in self-renew and cell cycle control in mES cells.In conclusion, we had established a high efficient lentivial RNAi system, working well in mES and other kinds of cells. After DUSP5, AIRE, and other genes had been interfered in mESCs, we suggested that DUSP5 and AIRE could be potential self-renewal genes. This study provided a new idea and data for understanding the molecular mechanism of self-renew regulation in ESCs. |
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