Study Of Construction Of Eukaryote Expression Plasmid PSG5-Oct14 And Its Instant Expression

ObjectivePluripotent and self-renewing state of embryonic stem cell(ES) makes itself become basic research and clinical application of seed cells. ES can be widely used on the premise of understanding its regulatory mechanism, studies reported maintenanceof pluripotency of ES inseparablely related with internal co-expression of some transcription factors. In particular, since 2006, induced pluripotent stem cells (iPS) have acquired remarkable progress, which prove that the transcription factors, such as Oct4,Sox2,Nanog, play a critical role in regulatory mechanism. In all of the transcriptional factors, Oct4 arouses general interest on maintaining pluripotency of ES. To date, it is not fully clear about regulatory mechanism of Oct4 gene.Fetal bone marrow mesenchymal stem cells (fBMSCs) are more plastic than adult BMSCs. Multi-directional differentiation has been considered as the most important biological characteristic of fBMSCs, which function is similar with ES. BMSCs can be induced into osteoblasts, cartilage, fat cells, nerve cells, islet cells and so on. Researches illustrate fBMSCs can express embryonic transcription factor Oct4. Because of ES is limited, we use fBMSCs to clone Oct4 gene, construct eukaryote expression plasmid pSG5-Oct4, transfection and instant expression of pSG5-Oct4 was detected in Mesenchymal cells derived fetal Pancreas. Try to transfect fBMSCs by pSG5-Oct4, so as to provide basis for the research of its biological function.Methods1,Mononuclear cells, used to isolate total RNA, were collected from bone marrow of abortion fetus aged 3-5 months by density gradient centrifugation.2,According to the restriction enzyme sites of pSG5 and Oct4 gene sequences(NM₀02701.4) reported in Genbank, we design primers to amplified open reading frame of Oct4 with PCR.3,The gene fragment of Oct4 and pSG5 are digested with restriction enzyme, then joined by T4 DNA joining enzyme.The chosen and identified recombination plasmid pSG5-Oct4 is transformated and the positive clone is with restriction endonuclease and sequencing.4,Mesenchymal cells derived fetal Pancreas were isolated by use of adherent culture method. Following 2-3 passages, cells were used to transfected.5,pSG5-Oct4(plasmid pSG5, or no plasmid, as control group) were transfected into Mesenchymal cells derived fetal Pancreas, the morphology and growth status of Mesenchymal cells were observed by inverted microscope.6,Whether Oct4 gene was expressed in Mesenchymal cells of fetal Pancreas was detected by RT-PCR, expression of Oct4 protein in Mesenchymal cells was detected with immunocytochemical staining.7,Try to transfect fBMSCs by pSG5-Oct4, observe morphological change of fBMSCs and detect with immunocytochemical staining.Results1,After RT-PCR reaction, gel electrophoresis shows a single bright band at 1.1 kb, the size of the fragment met the design expectation.2,DNA sequencing display the affinity was 97%to Oct4 gene(NM₀02701.4)in Genbank.3,After 24h, Mesenchymal cells derived fetal Pancreas were fusiform and showed clone cell-like.4 days later, more cells gathered into a bundle.9 days later, cells were covered the bottom.4,After 3 days transfection, Oct4-Mesenchymal cells gradually became round and changed into suspension cells.6 days later, more cells changed into suspension cells, some cells were agglomerate. While the control cells were still adhere and fusiform.5,gel electrophoresis show two bright bands were at 1.8kb and 1.2kb, the size of the fragment met the design expectation.The cytoplasmic of round Mesenchymal cells displayed the expression of Oct4 protein by immunocytochemical staining.6,After about 4 weeks transfection, some'fBMSCs changed into neurons-like cells the special markers NSE were examined by immunofluorescence.Conclusion1,In this study, we isolated and cultured mesenchymal cells derived fetal Pancreas.2,The eukaryote expression pSG5-Oct4 was successfully constructed by genetic engineering.3,pSG5-Oct4 could be transfected into mesenchymal cells derived fetal Pancreas, detected Oct4 protein by immunocytochemical staining.4,fBMSCs may be induced into neuron by Over-expression Oct4 gene, we should explore the truth of this phenomenon.