Expression Of Nanog Gene In Hela Cells And The Effect Of Its SiRNA On Mouse Embryonic Stem Cells

Embryonic stem cells (ES cells) derived from the inner cell mass (ICM) of blastocyst maintain two features: self-renew and differentiation. ES cells can differentiate into all types of cells. The molecular mechanism to maintain the pluripotency of EC cells was one of the main research focuses. Nanog gene is an impotent transcription factor maintaining the pluripotency of ES cells. It will help to understand the molecular mechanism in maintaining the pluripotency of ES cells that investigating expression mechanism of Nanog and the relationship among the Nanog, LIF/STAT3 and Oct3/4 in the pluripotency of ES cells. The homobox Nanog protein can maintain self-renewal of ES cells independed with LIF/STAT3 pathway. Nanog Oct4 and Sox2 are all impotent transcription factors maintaining pluripotency of ES cells and ICM. Nanog gene only expresses in ES cells and embryonic carcinoma cells (EC cells). It is undetectable in adult cells. In mouse ES cells, down regulation of Nanog gene by RNA interference can leads to the differentiation of ES cells into extraembryonic endoderm and activating the associated gene gata4, gata6 and lamininB1. Studying Nanog will enable us to investigate the essential pluripotential and self-renewal pathway of ES cells.In the experiments, Nanog gene was cloned from mouse ESCs and constructed Nanog expression vector. The cloned Nanog gene was expressed in Hela cells. The effect of Nanog expression on Hela cells was observed. According to the published sequence of Nanog gene, three pairs of siRNA fragments were designed, which were used to transfected into mouse ES cells and P19 cells.The project including two parts of experiment:â‘´According to the sequence of Nanog gene published in GenBank(XM₁32755,gi:20831594), I designed primers and cloned mouse Nanog gene (915bp) from mouse ES-D3 cells. DNA sequence analysis showed that the homology between Nanog gene sequence of mouse ES-D3 cells and the sequence publiced in GeneBank was 99.7%. The eukaryotic expression vector pG-Nanog was constructed by inserting the Nanog fragment into pEGFP-C1. The recombinant plasmid was transfected into Hela cells. The result of RT-PCR showed that Nanog gene successfully expressed in Hela cells. The stable transfected cells were also established with G418 selection. The results of the growth curve, PCNA staining and cell cycle assay showed that expression of Nanog in Hela cells enhanced cell proliferation and promoted cells to enter into S phase.⑵According to the sequence of Nanog gene, three pairs of siRNA fragment of N301,N749,N845 were synthesized. One unspecific siRNA fragment was also synthesized as the negative control. The siRNA fragments were transfected with Lipofectamine 2000 into ES-129 cells from 129 mice. For RT-PCR reaction, total RNA was isolated from ES cells after 24 hours transfection by TriZol method. The RT-PCR result showed that N301 was the best siRNA that significantly down regulate Nanog gene expression. ES cells from 129 mice and P19 cells from mouse embryonal carcinoma were transferred by Lipofectamine 2000 with siRNA N301 fragments. To form the embryoid body, after siRNA transfection P19 (1×104/ml) were grown by suspending cells in hanging drops for 48 hours. The results showed that N301 siRNA was efficiently interfered in Nanog gene expression in both ES and P19 cells. Down regulation of Nanog gene expression initiated ES cell differentiation and reduced the capability of P19 cell to form embryoid body.