The Impact Of Sidt2 Gene Knockout On Liver Gene Expression Of Mice

Part?Generation of Sidt2 knockout mice and liver phenotype studyObjectives To generate Sidt2 knockout mice and test the liver phenotype.Methods(1)Animal protocols were approved by the Shanghai Research Center For Model Organisms.Heterozygous(Sidt2-/+),wildtype and Sidt2(-/-)littermates mice were bred and genotyped.DNA from mouse tails have been amplified to confirm that sidt2 has been deletedat DNA level successfully.(2)Realtime PCR was used to detectsidt2 m RNA expression at liver tissue,and immunohistochemistry staining and Western Blot had been used to detect sidt2 expression at protein level.Results(1)Both the DNA and RNA level analysis suggested that the exon2 of the sidt2 gene had been knockout successfully(2)Western Blot and immunohistochemistry staining showed that distinct expression of sidt2 was observed in the control wide type mice but not in the sidt2 gene knockout miceConclusions Sidt2 knockout mice have been successfully generated to satisfy the following experiments.Part ? The impact of Sidt2 gene knockout on liver gene expressionObjectives To Screen differentially expressed gene profiles of liver tissue between sidt2 knockout mice and the control wide type mice with microarrays,and to investigate the mechanism of the liver lipid metabolism in Sidt2 knockout mice.Methods(1)Liver had been taken when the male sidt2 knockout mice and its control were 4,12 and 24 weeks of age.Screening of expression profiles were conducted with Affymetrix c DNA microarrays.(2)Real time quantitative PCR analysis was performed to verify part of differentially expressed genes.(3)Sidt2 knockout and control mice were fed high saturated fat(HF)diet for 14 weeks since the age of 8 weeks.Body weight and biochemical parameters(glucose,triglycerides,cholesterol,high-density lipoprotein,low-density lipoprotein,alanine transaminase,aspartate transaminase)were monitored.(4)Western blot has been applied to study the expression of leptin receptor and phos-stat3 protein in the JAK-STAT pathway.Results(1)There were 76 genes differentially expressed between sidt2 knockout miceand the wide ype mice based on the fold changes,e.g.more than 2 or less than-2.Among them,there were 12 genes correlated with lipid metabolism,while 15 of them were correlated with autophagy and apoptosis pathway.(2)Realtime PCR results were consistent with microarray data,indicating that c DNA microarray results accurately reflected changes in gene expression patterns in sidt2 knockout mice as compared with those of the wide type mice.(3)The TG level between these two groups that before and after the high-fat-diet were statistically significant.(4)The expression of leptin receptor in Sidt2 knockout mice was up-regulated,which sequentially activated the JAK-STAT pathway.Conclusions The knockout of Sidt2 gene in mice was associated with increases of plasma TG levels,this phenomenon may be realized by affecting the function of the JAK-STAT pathway.