Studies On Biological Functions Of MrgF And Agk By Using Gene Knockout Mouse Models

Part ?ROLE OF ORPHAN G PROTEIN COUPLED RECEPTOR MRGF IN NOCICEPTION STUDYThe gene knockout technology has been employed as the most efficient approach in multiple research fields of modern biology,including systematical gene function study,etiology of human disease,embryonic development research,and novel drug target investigation,as well.MrgF,an orphan G protein coupled receptor(oGPCR)is a member of Mas-related protein-coupled receptors family(Mrgs,is also known as sensory neuron-specific G protein-coupled receptors or SNSRs).Its ligands and biological function have been rarely reported so far.The mRNAs of most of the Mrgs family members are located specifically in small diameter sensory neurons in the trigeminal(TG)and dorsal root ganglia(DRG),which suggest Mrgs might play a role in pain transmission and modulation.Previously,the MrgF mutant mouse was generated in our lab.Behavior study results demonstrated that,compared with wild-type mice,MrgF-deficient mice exhibited similar response towards peripheral acute thermal and itch stimulus,but decreased sensitivity to capsaicin and formalin stimuli.The MrgF receptors are specifically expressed in cerebellar Purkinje cells and in sensory neurons in dorsal root ganglia,as well.The expression of c-fos and Penk,the pain related gene,were modulated by Creb phosphorylation in dorsal root ganglion.At the same time,the expression of Runx1,Nav1.7,Nav1.8 and Nav1.9 genes were significantly down-regulated in MrgF-deficient mice.This research work strongly suggested that MrgF play a critical role in nociception,which provided a better understanding on the biological function of Mrgs receptor family.Part ? ASSOCIATION STUDY ON AGK GENE AND MITOCHONDRIAL DISEASESMitochondrial disease is a group of disorders caused by dysfunctional mitochondria and ATP synthesis defect.It caused by mutation in the mitochondrial DNA and the nuclear DNA.Mitochondrial disease exhibited extremely variable phenotype and can present at any age.With the difficulties to diagnosis,few efficient treatments have been reported in clinical application.While numerous pathogenic mutations in both mt DNA and n DNA mitochondrial genes have been identified,the causal role of mitochondrial dysfunction in the common metabolic is still debated.However,to establish the mice model harboring gene mutations is permitting demonstration of the direct cause-and-effect relationship between mitochondrial dysfunction and disease.Sengers syndrome(MIM 212350)is an autosomal recessive disorder characterized by congenital cataracts,hypertrophic cardiomyopathy,skeletal myopathy,exercise intolerance,and lactic acidosis but normal mental development.Exome sequencing identified the causal nature of AGK mutation in Sengers syngrome.The molecular mechanism of AGK mutation in the mitochondria disease is still under investigation.Using conditional gene knockout technology,the Agk conditional knockout mouse model and the Agk full knockout mouse model were generated by crossing with EII?-Cre transgenic mice.Similar pathological phenotypes were observed in Agk-deficient mice in both skeletal and heart,compared with Sengers syndrome.Mitochondria structure abnormal and dysfunction of oxidative phosphorylation(OXPHOS)were also exhibited.However,unlike Sengers syndrome,no congenital cataract has been found in Agk-deficient mice model.In addition,mitochondrial membrane potential was evaluated in Agk-deficient mice MEF cells,which implicated that the abnormal apoptosis in cell might lead to origination of Sengers syndrome.Therefore,we established Agk knockout mice model to investigate the biological function of the gene at mitochondrial developmental stages and different tissues in vivo in order to provide essentially theoretical and experimental foundation for clinical screening of potential novel drug targets.