Experimental Studies On Establishing Mouse Embryonic Stem Cell Lines And Electroporating K14-EGFP Gene To The Mouse Embryonic Stem Cells In Vitro

Background: At present, the skin defect is one of the major problems encountered in clinical,how to make the tissue-engineered skin be used in clinical is the hotspot in tissue engineering research. In the study of the tissue-engineered skin,we need the type of seed cells which can be amplificated rapidly,have the ability of long-term culture in vitro and have exuberant cell function. Because embryonic stem cells have high regeneration and the potential to differentiate to other type of cells,so it is a good method to use the skin cells differented from embryonic stem cells to reconstruct skin. But at present, domestic and foreign scholars on the induced differentiation of embryonic stem cells to skin cells is not in-depth study.Objective: The study was designed to study and establish the mouse embryonic stem cell lines which include K14-EGFP gene fragment, as well as detect its characteristics.To lay a solid foundation for the basic research and clinical application of tissue-engineered skin.Methods: First, we establish the 129 / S1 mouse lines in vitro, and detect its characteristics though morphology,alkaline phosphatase staining,immunohistochemical staining of stage-specific embryonic surface antigen 1 (SSEA-1)and in vivo & in vitro differentiation. Then electroporation K14-EGFP gene fragment to embryonic stem cells, and also detect its characteristicsResults:1.Established the 129 / S1 mouse embryonic stem cell lines successfully, and detected its identification though the morphology,alkaline phosphatase staining,stage-specific surface antigen 1 and in vivo &in vitro differentiation.2.Ectroporated K14-EGFP gene fragment to embryonic stem cells successfully, amplificated the positive clones in vitro,so as to access to a large number of mouse embryonic stem which have K14-EGFP gene fragment.Conclusion:1. Establish embryonic stem cell lines from 3.5-day mouse blastocysts, and in the process of establishing ES cell lines MEF feeder layer and leukemia inhibitory factor are required so as to promote the cells to generate and inhibit its differente;2. The efficiency of electroporating embryonic stem cells is not high , but though the amplification of positive clones we can gain sufficient cells;3. When the ES cells ectroporated with K14-EGFP gene fragments are induced to skin cells which express keratin14,there will be the expression of green fluorescence;...