Research On Establishing Chicken Embryonic Stem Cell Line By PEGFP-hTERT Transfection

Embryonic stem cell is a kind of undifferentiated pluripotent stem cell, which was derived from ICM or primordial germ cell and contained all the genetic information. There were more and more studies on chicken embryonic stem cell lines building in recent years, and difficulty in making its cultivation is how to maintain their undifferentiated state while chicken embryonic stem cells proliferate indefinitely. Telomere is a section of DNA at the bottom of every chromosome in eukaryotic cell. Once every cell division, telomere is shorten50-100bp. When it is reduced to a certain value, the proliferation of the cells will stop proliferating, then enter into aging and apoptosis. Telomerase activation could be promoted by hTERT gene, so when the exogenous hTERT gene was transfeceted into cells, telomere shortening time will slow down or stop while cells proliferate. The eukaryotic expression vector pEGFP-hTERT was constructed successfully in this study, and transfected into chicken embryonic stem cells, which give rise to hTERT gene high expression within cells. Meanwhile through the improvement of the external culture conditions, the chicken embryonic stem cells are capable of proliferating for long-term stability, which could provide a theoretical basis and practical basis for the follow-up study of chicken embryos stem cell lines building. The main contents are as follows:1. Construction of eukaryotic expression vector pEGFP-hTERT and study on subcellular localization of its expression product in vitro. PCI-neo-hTERT was derived by plasmid DNA extraction and after that the hTERT gene was obtained by restriction enzyme digestion with EcoR I and Sal I.At last pEGFP-hTERT was built successfully through connected to the fluorescent eukaryotic expression vector pEGFP-N1by T4ligase. Then it was transfected into DF-1cells with the density of the5×105cells/mL inoculated into24well plates by LipofectaminTM,24h later, the pEGFP-hTERT were observed under fluorescence inverted microscope. Experimental group: cells transfected with recombinant plasmid pEGFP-hTERT; negative control group: cells transfected with pEGFP-N1; blank control group: non-transfected plasmid. The results showed there were expression of green fluorescence in the experimental group and the negative control group, which suggested that the hTERT gene located at the whole cell, indicating that the eukaryotic recombinant expression vectors were successfully constructed, with expression ability.2. The positive role of VC to promote chicken embryonic stem cell proliferation. Chicken embryonic stem cells were inoculated into96-well plates at the density of1×103cells/well, after that VC was added to the cell culture wells with the concentration of0μmol/L,10μmol/L,100μmol/L and1000μmol/L respectively, and the OD of each well was measured using CCK-8with microplate reader to detect cell proliferation at last. Since the OD value was proportional to the number of cells, the results showed that the OD value adding10μmol/L was higher than others, that was to say cell proliferation was the fastest, therefore the concentration of10μmol/L was the most appropriate for VC to promote the proliferation of chicken embryonic stem cells.3. The cESCs were transfected with pEGFP-hTERT and subcultured. The cESCs were isolated with tweezers from blastoderm of the stage X embryos of chicken, then cultured with80%BRL-CM and20%DMEM with exogenous cytokines added proper concentration of VC. Afterwards cESCs were identyfied by alkaline phosphatase (AKP) activity, stage-specific embryonic antigen-1, SOX2and OCT4. the result showed that all of these were positive, which indicated the maintenance in the undifferentiated state, and then the cESCs could be used for following inducing experiments. After that, the cESCs transfected pEGFP-hTERT were filtered by G418at the concentration of300ng/μL to obtained positive cells, then Quantitative PCR was operated to detect the expression levels of genes related to cell sternness. The final results showed that cESCs transfected pEGFP-hTERT could be passaged to15generations continuously, and stem cell specific surface antigen test were totally positive, as well as there were no significant differences in the expression levels of genes related to cell sternness.