The Expression Of FoxO1 Mutant Gene In Vitro And In Vivo

Metabolic syndrome including obesity, diabetes, hypertension and cardiovascular and cerebrovascular diseases, seriously harm to human health. The studies show that Foxo1-involved leptin resistance plays an important role in the pathogenesis of metabolic disorder, the interaction of Foxo1 and its target gene becomes one of the research focuses in the field of energy metabolism. FoxO1 can act on the promoters of neuropeptide POMC gene, inhibiting the transcription, thereby blocking the signal pathway of leptin and affecting energy metabolism of animals. In order to study the role of DNA binding domain(DNA Binding, Domain, DBD) in the function of FoxO1-mediated inhibition, the laboratory constructed a mutant FoxO1 with the DBD deleted, in plasmid p CMV5-Myc-FoxO1Δ4-5(p FΔ4-5). On this basis, in vitro and in vivo expression and functional studies was carried out in this thesis. First of all, plasmid p FΔ4-5 was amplified and analyzed by the means of enzyme digestion and DNA sequencing; then the plasmid was transfected into 293 Rb cells(293 cells containing the leptin receptor). The result of western blot showed that the target protein was detected, indicating the expression vector was constructed successfully; The results of luciferase assay showed that intact FoxO1 in the presence of DBD inhibited the activity of POMC promoter, however, FΔ4-5 without DBD not only lost the inhibition function, but significantly enhanced POMC promoter activity, indicating that DBD is required for FoxO1 to inhibit POMC gene; finally, the mutant gene was microinjected into zygotes to generate transgenic mice. 6 F0 transgenic mice were obtained and crossed with wild type mice for the passage of generations. Genotyping results showed that each of 6 F0 transgenic mice had positive offspring, indicating that the mutant gene integrated into the genome of transgenic mice. Subsequently, the expression of transgene was detected in hypothalamus, liver, white fat, brown fat, spleen, heart, lung and kidney by western blot. However, results showed that the target protein in transgenic mice were undetectable in all the tissues above, suggesting the transgene was somehow silenced. In order to determine whether the silence of target gene is due to the promoter methylation, we investigated the methylation status of CMV promoter in liver of transgenic mice genomic by bisulfite sequencing method. The results showed that 7 Cp G on CMV promoter had higher degree of methylation as we predicted. Then the demethylation of CMV promoter was tried by the intraperitoneal injection of 5-aza with different doses. The results of methylation analysis of CMV promoter and western blot of the target protein showed that although treatment of 5-aza demethylated the promoter to some extent the expression of the transgene was not detectable. Conclusion: 1. Results of in vitro experiments showed that DBD is required for FoxO1 to inhibit POMC gene; 2. CMV promoter can drive target gene expressing efficiently in cultured cells 293Rb; 3. FoxO1 mutant gene silencing in transgenic animals may be initiated by the CMV promoter methylation.