The Expression Of FoxO1 Mutant Gene And Phenotype Analysis In Transgenic Mice

Leptin is an important factor in regulating animal energy metabolism derived from adipose tissue and its function in central is to restrain the appetite of animals and promote the animal’s energy consumption.Leptin applys to the leptin receptor in hypothalamus and activate OB-Rb signaling pathways to regulate physiological fuction.In the case of leptin resistance,leptin signaling pathway is blocked,animal performance for obesity and other metabolic disorders.A study shows that the FoxO1(Forkhead box O1)increased of animals in the hypothalamus is one of the reasons leading to leptin resistance.The laboratory has carried on the preliminary study for the function of different FoxO1 mutant.One of the Fox01 mutant which knocked out the DBD(DNA Binding Domain)can promote the effect of leptin signaling pathway in cell experiments.In order to validate its role in the animal,laboratory builded a plasmid named p EF1α-Myc-Fox01 Δ DBD and got a total of about 10 mutant Fox01 transgenic mice founders using microscopic injection which background is C57 BL / 6.The purpose of this paper is mainly detection of gene expression in transgenic mice,tissue distribution and its impact on animal physiology.First,we carried on the genotype identification of genetically modified mice,make the 10 transgenic mice cross with wild type C57 BL / 6 to get a bigger group.To established a reliable method of genotype identification,the author changed the PCR system many times to overcome the purpose gene amplification because the gene has a high GC content and isnot easy to amplification.At the same time,to overcame genotypes identified the problem of easy pollution,avoid the occurrence of the false positive results,the author change gloves,clean and burn scissors continuously in the process of collecting samples of rat tail,and use pipette tips which has a filter net to add sample.According to this method,the authors have identified transgenic mice frome F0 to F5 generation,of which 8,9 founder cannot reproduce,line 1,2,3,4,5,6,10 series of genetically modified mice according to the experimental results of follow-up,do only a small amount of storage,line7 transgenic mice as experimental object remains around 500;Second,using the method of fluorescence quantitative PCR detect the target gene expression levels of the organisations in transgenic mice.Only line 7,according to the results of the liver of mice purpose gene express high levels;In the brain only line 3,7 mice have a high level of gene expression;There is only line 7 in white adipose tissue of mice have a high level expression of gene,line 3 mice have a low level gene expression.In the hypothalamus line7 mice have high level expression,line2,3 purpose gene in mice have medium level expression,line 1,10 mice have low gene expression;In brown adipose tissue and gastrocnemius line 7 mice expression of gene in also have a high level;Third,using Western Blotting detect target protein expression in hypothalamus in line 7 transgenic mice.Results show that the level of target protein expression is low;Fourth,detection of body weight and food intake of line 7 mice.According to the results,The positive mice weight is slightly lower than negative mice but the difference is not significant,food intake of mice did not differ significantly in mice,but the ratio of food intake than weight,show significant differences and the positive mice is greater than negative mice;Fifth,using quantitative fluorescence quantitative PCR and Western Blotting detect suppressing appetite POMC(Proopiomelanocortin)neuropeptide and promote appetite AGRP(Agouti gene-related protein)m RNA expression of leptin signaling pathways downstream and leptin sensitivity line 7 mice.According to the results,under the conventional condition POMC and AGRP expression have no significant difference,for a period of time in mice of jugular vein injection of mice leptin and effect after a period of time,detected positive mice STAT3(Signal transducer and activator of transcription 3)phosphorylation levels higher than the negative mice,while the POMC expression of experimental group compared to control group were not significantly increased;Sixth,using fluorescent quantitative PCR detect UCP1-3(Uncoupling protein 1-3)expression in brown fat in line7 mice.Results show that the UCP1 expression in positive mice in increased significantly(P < 0.01),UCP2,UCP3 have no significant change.