| Animal models are one of the most important tools for studying human diseases,gene function,diagnosis and treatment.Miniature pigs are more similar in metabolism,physiology,anatomy and organ morphology with human than other animals(such as mouse).In addition,the reproductive performance of pig shows shorter cycle and more fertility.Therefore,the use of mini pigs to produce human disease models,drug development,and xenotransplantation shows good prospects and advantages.ENU(acetylnitrosourea)is widely used for inducing mutation to obtain disease models such as in zebrafish and mouse.ENU affects DNA replication by directly infiltrating into spermatogonia,and induces genomic mutations in sperm.In this study,ENU mutagenesis was performed on Large White and Bama pigs,and the phenotype about metabolic disease was screened in the Generation F1.In the experiment,the ENU injected boars were mated with wild type pigs to produce Generation F2,and Generation F2 sows were backcrossed with their fathers to obtain mutant Generation F3.The metabolic disease-related mutant individuals were screened by blood routine and biochemical indicators.A total of 18 Large White pigs families were established,and 1236 mutants in Generation F3 were obtained and used to screen biochemical indicators.For Bama pigs,a total of 30 families were established,and 1838 mutant pigs in Generation F3 were obtained and used to screen biochemical indicators.The results showed that there was no significant difference between the mutant groups and control groups in body weight,body length and body height(p>0.05),but biochemical indicators of 47 F3 Large White pigs and 56 Bama minipigs were significantly different with control pigs(p<0.05).On the basis of mutagenesis,we focused on the screening of potential metabolic disease F3 individuals with high blood lipids,high cholesterol,abnormalities in the heart,blood vessels and so on by high-fat diet induction.24 one-month-old Bama miniature pigs were randomly divided into control group and high-fat induction group.The control group used normal diet.The high-fat induction group was fed with 2.0%cholesterol feed for 6 months.Body weight,blood routine and biochemical indicators,coronary angiography,histological examination of various organs and liver transcriptome analysis were performed after high-fat induction.The body weight of the high-fat induction group was significantly higher than that of the control group after 4 month induction,and the blood triglyceride was significantly higher than that of the control group after 6 months induction.Abnormal coronary angiography and tissue section of each organs were not observed in high-fat induction group.The results of liver transcriptomic analysis showed that there were 2468 differentially expressed genes between the high-fat-induced group and the control group.The differentially expressed genes were mainly related to the metabolic pathway.By deep analysis,we found the expression of genes involved in insulin signaling pathway was inhibited(including),and the IRS-2 gene was significantly lower in the high-fat-induced group than in the control group,indicating IRS-2 plays important role on lipid metabolism.IRS-2 is an important signaling substrate in the insulin signaling pathway.In this research,we attempted to study the function of IRS-2 in pig in order to determine whether IRS-2 could be used as a target gene for establishment of porcine metabolic disease model.Two fragments(153 bp and 427 bp)of porcine IRS-2 gene were amplified based on homologous sequence among different species,and 5’/3’RACE was performed to obtain a full-length cDNA.The full-length cDNA of porcine IRS-2 is 5816 bp,including 214 bp 5’ untranslated region(UTR),3276 bp CDS region,and 2323 bp 3’UTR.Then,we detected the expression pattern of IRS-2 mRNA in each tissues of pig.We found IRS-2 mRNA was detected in all the tissues examined.IRS-2 mRNA was highly expressed in the cerebral cortex,hypothalamus,cerebellum,and liver,and lowly expressed in the jejunum,kidney,heart and pancreas.The IRS-2 protein was highly expressed in the cerebral cortex,cerebellum,and hypothalamus;on the contrary,it was hardly detected in the jejunum,kidney,heart,and pancreas.Overall,IRS-2 protein mRNA expression patterns were consistent among the tissues.Immunohistochemical analysis showed that the location of IRS-2 was localized in the arcuate nucleus,the ventromedial nucleus,and the paraventricular nucleus of the hypothalamus.In the cerebral cortex,IRS-2 also showed positive staining.This research performed functional studies of IRS-2 in porcine hepatocytes and aortic vascular endothelial cells by RNAi.In hepatocytes,IRS-2 knockdown resulted in a significant increase in the expression of PEPCK,SREBP-1 and LXR mRNA,while GCK mRNA was significant decreased and Fbp-1 mRNA was no significantly changed.We further tested the genes related to cholesterol metabolism downstream of LXR.The results showed that the expression levels of ABCG8 and CYP7A1 were significantly increased in IRS-2 knockdown cells.Subsequently,we evaluated the changes of HO-1,VEGFA,VCAM-1,GLUT-1,and GLUT-2 after IRS-2 knockdown on aortic endothelial cells.The results showed that the expression levels of eNOS and HO-1 mRNA were significantly decreased.In contrast,VCAM-1 mRNA expression was significantly increased in IRS-2 KD cells.The expression of VEGFA,GLUT-1 and GLUT-2 mRNA did not change significantly after IRS-2 knockdown.Furthermore,in order to establish an IRS-2 knockout animal model,we obtained IRS-2 knockout porcine fetal fibroblasts using the CRISPR-Cas9 gene editing system and IRS-2 knockout cloned embryos.The study laid the foundation to establish porcine metabolic disease models.To sum up,conclusions of this study can be reached as follows:1.Individuals of metabolic abnormality Generation F3 were obtained by ENU mutation;2.The complete cDNA sequence of pig IRS-2 was cloned successfully,and the expression pattern of porcine IRS-2 in different tissues was determined;3.It was clear that knockdown of IRS-2 could lead to abnormal function of porcine hepatocytes and arterial endothelial cells.These results will help to establish a diabetic and vascular related porcine metabolic disease model by targeting IRS-2. |
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