SiRNAs Interference The Nanog Gene Of ES Cells And The Eukaryotic Expression Vector Construction Of Nanog

RNAi exists in all the cells of organism, whose molecular mechanism has been known theoretically, and provides a new method for studying the stem cells. The technological research on RNAi had great prospect in studying the function of genes and the maintenance and specific differentiation regulation of stem cells. On the study of the ES cell pluripotency and the specific differentiation, the homeoprotein Nanog is the only known factor, which is capable of maintaining ICM and ES cell pluripotent independently of LIF/Stat3. Nanog is expressed in the early embryonic cells and ES cells mostly and has a relationship of time and order with the LIF/Stat3 and Oct4, which are also expressed in the early embryonic cells and ES cells. It will help to understand the molecular mechanism in maintaining the pluripotency of the ES cells that investigate the relationship between the expression mechanism of the Nanog and the function of the LIF/STAT3 and Oct4 in the potential of the ES cells. In the research, four transient interference vectors and a stable one aimed at the mouse nanog gene were constructed so as to study the change of the cytobiology of the ES cells by interfering the expression of the nanog gene with the RNAi technology. Also, a recombinant expression vector had been constructed that contained the whole open reading frame of the nanog gene of the mouse from the initiation codon to the stop codon. This is a basic work for the next step to study more of the molecular mechanism of the nanog gene in maintaining the pluripotency of the ES cells by combining the adult differentiated cells expressing the exogenous Nanog and the ES cells being interfered the nanog gene. The work consists of five parts as follow: 1. Cloned and sequenced the nanog gene from the mouse ES-D3 cells with the primers nanogB and nanogC. Analysis with the Align Software showed that the homology was 99.5% between the nanog gene sequence of the mouse ES-D3 cells and the mouse ES cells (XM₁32755, gi: 20831594) in GenBank. It showed that it was feasible to study the function of the nanog gene in the ES cells by using the mouse ES-D3 cells. 2. Observed the Nanog expression level and the growth behavior of the mouse ES-D3 cells cultured on the MEF feeder, the neonatal bovine testicular sertoli cells (nBTSCs) feeder and the neonatal rabbit testicular sertoli cells (nRTSCs) feeder on the 4th day. Semi-quantative RT-PCR showed that the ES-D3 cells cultured on the MEF feeder expressed more Nanog on the 4th day than those cultured on the other two kinds of feeders. Results showed that the colony forms and the differentiation degree of the ES-D3 cells cultured on the different feeders were different. Colonies of the ES-D3 cells cultured on the MEF feeder showed very regular form and strong positive for AKP staining. The cells were compact and there were few big cells on the surface of the colonies. Colonies on the nBTSCs feeder had clear edge but spread some big cells on the surface. Most cells in the colonies showed positive for AKP staining and a few showed weak positive. Colonies on the nRTSCs feeder were irregular and unclear. Only part of the colonies did show positive for AKP staining. There were a few big cells on the surface of the colonies and the fibroblast spread around them. 3. Selected 4 regions of the nanog gene of the ES-D3 cells to synthesize the oligonucleotide and constructed four siRNA transient expression vectors which containing the murine U6 promoter to transfect the mouse ES-D3 cells. Analysis by the semi-quantative RT-PCR showed that 3 out of the 4 siRNA expression vectors could translate the siRNA or shRNA in cells and could induce RNAi to inhibit the expression of the target gene nanog. After 48 h of the interference for ES-D3 cells, we found that although the form of the colonies had little direct difference between the interference cells and the un-interference cells, but the former was less upheaved and more irregular formation than the later. The growth speed of the interference cells was also slower. The positive intensity of the AKP staining had no difference between the interference cells and the un-interference cells. 4. With the basis of the transient recombinant interference vector pHUsi305, which had the best interfering effect, we had constructed a stable interference vector named pGsi305 that brought a reporter gene and could screen the stable cell strain the nanog gene was interfered. This is a basic work for the next step to study more of the molecular mechanism of the nanog gene in maintaining the pluripotency of the ES cells. Analysis by the semi-quantative RT-PCR showed that the DNA band of the nanog gene of the ES-D3 cells transfected with pGsi305 was weaker evidently than the bands of the cells transfected with pEGFP-C1, the untransfected cells and the endogenous control ?-actin. It showed that the pGsi305 could inhibit the expression of the nanog gene effectively. Passaged and then screened with G418 after transfecting the pGsi305 and the pEGFP-C1 for 48 hours. The ES cells transfected the two plasmids had still evident colonies at the 7th day. And at the 14th day, colonies transfected with pGsi305 began to become relaxed, but a few of the cells had still maintained their pluripotency. The colonies showed weakly positive for AKP, c-kit and Oct staining, but the SSEA-1 was negative. At the 18th day, the ES cells transfected with pGsi305 had no visiblecolonies already and only could find a few of differentiated cells with fluorescence, and all of the cells showed negative for the AKP, c-kit, Oct4 and SSEA-1 staining. On the contrary, except for the descent vitality, the ES cells transfected with pEGFP-C1 still had typial colonies at the 14th day and the 18th day. 5. A pair of expression primers containing Bgl II and Sac II at the 5'-end respectively were designed to amplify the whole open reading frame of the nanog gene of the mouse ES-D3 cells containing 305 amino acid. Ligated the target gene with the clone vector pMD-18T and transformed the ligation product into the E.coli JM109. Cultured the positive colonies and then extracted the recombinant plasmids pMD915. The pMD915 and the pEGFP-C1 were digested with Bgl II and Sac II respectively to produce the cohesive ends in order to ligate the target gene into the pEGFP-C1 to get the recombinant eukaryotic expression vector pG-bnanog. The insert position, the orientation, the size and the ORF were right by PCR, digestion and the sequence analysis.