Generating DXPR1-GFP Fly And The Location Of DXPR1 In Drosophila Larval Central Neuronal System

Primary familial brian calcification(PFBC)is a rare neurodegenerative disease.It is a common chromosomal disease presented familial inheritance and characterized by calcification in brain tissue.It is also called idiopathic basal ganglia calcification(BGC).Calcification can also occur in the caudate nucleus,the bean nucleus,dentate nucleus,and the thalamus.The onset age of the patients is between 30 and 60 years old,the clinical manifestations were characterized by dystonia,multiple seizures,cognitive dissonance,chorea,athetosis,or Parkinson's disease.So far four genes,SLC20A2?PDGFB?PDGFRB and XPR1,were known as PFBC disease causing genes.Recently identified PFBC causing gene XPR1 was initially identified as the cell surface receptor for xenotropic and polytropic murine leukemia retroviruses(X-and P-MLV).Bioinformatics methods were used to predict the XPR1 protein figure.XPR1 is an eight times transmembrane protein and contains two evolutionary conservative domains,SPX and EXS.The mutations of XPR1 in most PFBC patients were located in the SPX domain and presumably it's function as phosphorus transporter.The EXS domain may be responsible for phosphate concentration perception.XPR1's homologous gene is CG10483 in Drosophila melanogaster,located in the chromosome 3.CG10483 has high homology with human XPR1,and especially in SPX domain.This suggested that fly is the reliable system to study the function of the XPR1 gene and its role in the phosphate balance.Due to the dXPR1 antibody prepared in our previous studies can only be used in western blot experiments and the background is very higher in immune staining.In order to observe the location of dXPRl in vivo of flies,we try to generate endogenous expression dXPR1-GFP flies to research on the location and function of dXPRl proteins.In our researches,we used the CRISPR/Cas9 method to generate the endogenous dXPRl-GFP fusion protein expression flies.First,the specific expressed Cas9 enzyme's flies were cross with the flies expressed sgRNA.The vector of HDR donor was microinject into the eggs of the crossing.The dXPR1 gene target site can be split by Cas9 enzyme and the dXPRl-GFP gene was produced by the homologous recombinant repair.Through genetic screening,we have successfully obtained fruit flies expressing dXPRl-GFP fusion protein.In the direct observation and immunostain,the location and expression level of the fusion protein in the brain of the third instar larvae was observed.The dXPR1-GFP was mainly located in the mashroom body and ventral ganglion of third instar larval brain.The flies will provide effective tool for the functional study of dXPRl and also for exploring the role of dXPRl in phosphate homeostasis.