Construction And Analysis Of DEPTOR Gene Knockout Mice Model

Research BackgroundDiabetes melitus is a clinical syndrome characterized by blood glucose disturbance caused by the interaction of genetic factors and various environmental factors.Mammalian target of rapamycin(mTOR)was found to be an evolutionarily conserved serine/threonine protein kinase.Studies have shown that mTOR participate in the pathogenesis of diabetes by affecting the pathogenesis of insulin resistance through various pathways such as insulin signaling pathway and pancreatic beta cell generation.DEPTOR is an endogenous mTOR inhibitor.Studies have found that DEPTOR also has an important influence on cell metabolism.Up to now,there is little research on whether DEPTOR plays a role in the pathogenesis of diabetes,especially the study of islet beta cells.Technology of gene knockout is mainly used in the functional research of the target gene.The Cre/Loxp system is the most widely used conditional gene knockout technique.In this study,DEPTOR was used as a target gene to explore the role of DEPTOR in the pathogenesis of diabetes,especially in the aspect of pancreatic β-cells,thus providing important information for the therapeutic intervention of diabetes and β-cell failure.Research Method1.Observe the expression of DEPTOR in islet cells in db/db diabetic mouse model.2.According to the principle of Cre-loxP system,a mouse model of conditional whole body knockout DEPTOR gene was constructed.3.According to Cre-loxP system principle,a mouse model of knock-out DEPTOR gene specific for pancreatic islet beta cells was constructed.4.To verify the knock-out effect of conditional knockout of DEPTOR gene mouse model.5.To verify the knock-out effect of conditional knockout ofbeta cells specific knockout DEPTOR gene mouse model.6.Western blot,immunohistochemistry and immunofluorescence were used to detect the phenotypic changes in knockout mice.Statistical AnalysisIn this study,all data were replicated for 3 times or more,and the results were represented by mean±standard error and analyzed by t-test,and two-way ANOVA.P<0.05 was considered as statistically significant.Research Results1.The expression of DEPTOR gene was significantly increased in pancreatic beta cells of db/db diabetic mouse model.2.The model of the conditioned DEPTOR gene was successfully constructed.Compared with the wild type(WT)mice,the Western blot results showed that the expression of DEPTOR in the knockout mice tissues decreased significantly.2.The conditioned mouse model of the islet beta cell specific knockout DEPTOR gene was successfully constructed.The results of Western blot showed that the expression of DEPTOR in the pancreatic tissue of knockout mice was significantly decreased.The results of double immunofluorescence showed that the expression of DEPTOR in the islet beta cells of the knockout mice decreased significantly as compared with the control group.4.Preliminary results in this study showed that there was no significant difference in the number and morphology of islet beta cells between the conditional DEPTOR knockout mice and the control mice.Conditional pancreatic beta cell knockout of DEPTOR mice showed impaired glucose tolerance in the abdominal glucose tolerance test.ConclusionThe conditional DEPTOR gene knockout mouse model was successfully constructed and the conditioned DEPTOR cell knockout mouse model was constructed for the first time.The preliminary exploration of some phenotypes was carried out,and the research platform for further study of DEPTOR gene in the pathogenesis of diabetes was also provided.