Identification And Phenotype Analysis On Disruption Of CASK Gene In Mouse Pancreatic β Cells And The Regulation On CASK

Part 1:Identification and phenotype analysis on disruption of CASK gene in mouse pancreaticbcellsObjective:Diabetes mellitus is a type of metabolic disease characterized by elevated blood glucose.The calcium/calmodulin-dependent serine protein kinase（CASK）is involved in intracellular and extracellular signal transduction,gene transcription regulation in the nucleus,transport and release of neurotransmitters and signal transduction in the postsynaptic membrane.Our previous studies showed that CASK was involved in the anchoring of insulin vesicles.In order to verify the experimental results in mouse and study the effect of CASK gene deletion on insulin vesicle transport,a mouse model of specific knockout of the CASK gene in pancreatic isletβcells was established in this study.Preliminary identification and phenotypic analysis were performed.Methods:1.Using conditional gene targeting and chromosomal site-specific recombinase Dre-Rox system,two homologous loxP sites were placed on both sides of exon 6 of the coding key domain of the CASK gene by homologous recombination to obtain a normal phenotype of CASK^loxP/-mice.2.After hybridization between CASK^loxP/YoxP/Y mice and MIP-Cre/ERT mice,we obtained isletβ-cell CASK-specific knockout mice.3.Immunofluorescence validates CASK knockout efficiency.Results:Southern Blot showed that the CASK^loxP/-mice had the correct gene tag,and immunofluorescence showed that CASK was found in various cells of the pancreas.Conclusion:The pancreatic tissue-specific CASK knockout mice were successfully prepared.The phenotype of isletβ-cell CASK-specific knockout mice was normal.Part 2:Transcriptional regulation of CASK by transcription factor PPARγObjective: To investigate the transcriptional regulation of CASK by transcription factor PPARγ.Methods: Changes in insulin secretion were observed in islet β cell line INS-1 cells treated with different concentrations of rosiglitazone;INS-1 cells were treated with rosiglitazone at different concentrations and at different times;quantitative PCR,western blot,and immunofluorescence were performed to detected the expression of CASK;chromatin immunoprecipitation was performed to search for a binding site for the transcription factor PPAR gamma in the CASK promoter region;the effect of rosiglitazone on CASK was observed after pretreatment of INS-1 cells with PPAR gamma-specific interference fragments;when the expression of CASK was disturbed,the effect on rosiglitazone-stimulated insulin secretion was not influenced.Results: The KSIS function of INS-1 cells was significantly enhanced after rosiglitazone treatment（P<0.05）.The m RNA and protein levels of CASK in INS-1 cells treated with rosiglitazone increased in a dose-and time-dependent manner.（P<0.05）;chromatin immunoprecipitates showed that PPARγ has a binding site in the CASK promoter region;rosiglitazone modulates CASK partially through the PPARγ pathway（P<0.05）;There was no effect on rosiglitazone-stimulated insulin secretion when the expression of CASK was disturbed（P>0.05）.Conclusion: 1.PPARγ agonist rosiglitazone upregulates CASK gene expression in INS-1 cells.2.CASK does not participate in the regulation of insulin secretion by rosiglitazone.