Establishment Of Nanog-EGFP Mouse Embryonic Stem Cell By CRISPR/Cas And Screening Of T He Effective Ingredient In Culture Medium

Embryonic stem cells (ESCs) have become powerful tools for the study of gene functions, cell therapy, tissue regeneration, etc., by it’s self-renewal characteristics and pluripotency in vitro. In recent years, the technique with ESCs was rapidly developed and contributed to the breakthrough for clinical researches so that the status of ESCs was much regarded by scientists in the field of regenerative medicine. In the year of 1981, scientists achieved the isolation and culture of mouse embryonic stem cells in vitro with feeder layer culture system successfully. As to ESCs culture systems, which had experienced a long period. N2B27+2iL, a sort of culture system, was widely applied to mice ESCs culture from 2008, which has become the preferred alternative for most laboratories to culture mouse embryonic stem cells. Athough this culture system can maintain the clonal morphology and pluripotency of mouse embryonic stem cells in vitro completely, the complex ingredients and the higher costs makes this culture medium impractical for routine use. It is an important issue to simply and define the ingredients of this medium so as to reduce experimental benefit-cost ratio without lowering culture effect.With the development of gene editing technique, multiple scientific areas have made a great progress especially in stem cell researches. From ZFNs to TALENs even to CRISPR/Cas9 and, from the initial identification for DNA sequence by proteins toRNAs, the development of gene editing technology is increasingly amazing. In recent years, the innovative CRISPR/Cas9 gene editing technique pushes gene editing researches forward to a new era. This technique relies on sgRNA recognition to the specific locus and DNase activity of Cas9 protein, which result in DNA double-strand breaks. Subsequently, homologous recombination is triggered off for gene repair, which improve the efficiency of gene knock-out and genetic recombination considerably. Thus, it provide a new technical platform for scientific studies.On this dissertation, Nanog-EGFP mice ESCs line has been constructed by CRISPR/Cas9. and we regarded it as a reporting system which can be used to screen effective ingredients of N2B27 medium. Analyzed by fluorescence microscope, flow cytometry, and HCS analysis system, we identified some certain ingredients of N2B27 which have played important roles in maintaining the pluripotency of ESCs The result showed that the concentrational decrease of insulin, transferrin, progesterone, putrescine and sodium selenite was harmful for pluripotency maintaining. Meanwhile, neurobasal as a basic culture medium might associate with one of five ingredients to maintain clonal morphology. Also, this dissertation not only provides a new technique platform and research strategies for mice ESCs studies, but gives instructive significance to clinical applications.