The Effect Of Gadd45g On The Self-renewal Of Mouse Embryonic Stem Cells

Mouse embryonic stem cells originally isolated in laboratory conditions in 1981, the cell line is the first totipotent embryonic stem cell lines. Based on the method of establishing mouse embryonic stem cell line, scientists have isolated embryonic stem cells constantly at different livestock animals and to establish the appropriate system. Because embryonic stem cells are in possession of the unique biological properties comprising self-renewal and pluripotency, they have a very important significance both from the perspective of developmental biology and in the clinical research. For a more thorough study about the mechanisms that affecting embryonic stem cell-related characteristics, more researchers will study focused on how to maintain their state of self-renewal in vitro; and how to induct them make a directional differentiation, in order to establish a model for clinical disease and so on. With further research, scientists have developed the methods that can maintain embryonic stem cells state in vitro. However, to maintain the self-renewal of embryonic stem cells is a more complex biological process, the effects of different genes, different paths or different external conditions all will change its state. Currently researches in some embryonic stem cell-related genes, pathways and regulatory mechanisms are not perfect, which hinders its breadth in the application. So study the new gene which impact on embryonic stem cells by means of molecular experimental techniques, and facilitate amore comprehensive understanding of mechanisms that regulate embryonic stem cells maintain self-renewal and differentiation.Gadd45g, as our studytarget, is a new gene for research of embryonic stem cells, there is come so far no related report has claimed that this gene can react on mouse embryonic stem cells to maintain it’s self-renewal. There are a large number of reports indicate that Gadd45g is associated with tumorigenesis, come so far it has been confirmed related to pituitary tumor, meibomian adenocarcinoma, squamous cell carcinoma, colon cancer and other tumor cell’s formation’’. And we have already known that there are many similar biological characteristics between stem cells and cancer cells, so we supposed that Gadd45g may have some effects on embryonic stem cells too, and boldly presumed it might be associate with proliferation of embryonic stem cells. We want to explore the role of Gadd45g in mouse embryonic stem cell self-renewal process through this project research, first we need to make overexpression of the gene in embryonic stem cells, while design the control group for comparison analysis. Designing primers of the gene with methods of molecular biology experiments and make a large number of amplification, after sequencing to verify correctness, introducing the gene into 46C cells with assistance of liposomes, overexpressing and testing the result. When stable cell lines has been established, make full use of characteristics of embryonic stem cells, such as in the state of self-renewal, embryonic stem cells can be stained by alkaline phosphatase while differentiated ones can’t be colored, we can judge the state of the cells by this way. Through reading a lot of literature, we can filter out some specific markable genes, such as embryonic stem cells maintain self-renewal stage’s markable gene Oct4, Nanog, and so on, with the help of immunofluorescence, using a secondary antibody with dye to identify it’s corresponding primary antibody, which can depending on the relative content of fluorescence under microscope to deduce the expression of the corresponding protein, thereby determining embryonic stem cells in which growth state. Experimental results show that after overexpression Gadd45g, the growth rate of embryonic stem cells decreased obviously, cells demonstrated a significantly differentiated state, indicating Gadd45g has good effects on inhibiting embryonic stem cells’self-renewal and promoting their differentiation. After the initial determination, we would like to use some functional verification experiments to explore its role. First, we chose to do qPCR experiment, determining cell’s stage by marker genes and meanwhile by means of expression change of marker genes in different developmental state of cells to infer Gadd45g may affect which specific stages of embryonic stem cell development. We screened Esrrb, Tlcp2ll, Soxl7, Gata6, Foxa2, Mixll, GSC,Nestin, Soxland other marker genes, comparative analysis of the results shows that overexpression Gadd45g plays a greater influence about the expression of mesodermal marker genes and endoderm marker gene, it can be inferred that Gadd45g has strong performer on the stage of the development of mesendoderm.To verify the expression quantity of its protein, we also did a Western Blotting experiment. Because of there has been some previous reports indicated that the path play a role in LIF/JAK/STAT3 signaling pathway, we also chose STAT3 to verify as a target protein. Through analysis of experimental results, we found that STAT3 phosphorylation expression of experimental group, overexpression Gadd45g, shown a significant change compared with the control group, it can be inferred the gene also affected LIF/JAK/STAT3 signaling pathway in embryonic stem cells. In the early time there were a large number of literature had reported some small chemical molecules can play a role in maintain embryonic stem cells self-renewal, and during the experiment, we observed that after overexpression Gadd45g, the growth rate of embryonic stem cells had a clear downward trend compared with the control group cells, based on this we screened a large number of small molecules, such as CHIR, PD03, ActivinA, bFGF, etc; trying to find ways to suppress the effect of Gadd45g on embryonic stem cells. After culturing in different conditions, cell counting alkaline phosphatase staining, we selected Y27 that can suppress the impact of Gadd45g on embryonic stem cells.A mass of analysis about experimental data indicate that Gadd45g has a certain role in the proliferation of mouse embryonic stem cells, it can suppress self-renewal and promote differentiation; we also found that it can affect the process of the development of mesendoderm in embryonic stem cells and regulate LEF/JAK/ STAT3 signaling pathway. However, at the present there are not yet relevant reports about the gene’s effection on embryonic stem cells. Therefore, the study of this subject found a new gene to inhibit embryonic stem cells maintain self-renewal, enriched this gene’s effection and also expanding the research range of embryonic stem cells.