Generate A DPit:GFP Fly Stock With The CRISPR/Cas9 System

Familial Idiopathic basal ganglia calcification(IBGC)in humans causes brain calcifications.These calcifications typically occur in the basal ganglia,which controls the human locomotion.The clinical features of IBGC are locomotion disorders and emotional/behavioral difficulties.The locomotion disorders in IBGC include involuntary tensing of muscles,uncoordinated movements and uncontrollable movements of the limbs.The psychiatric or behavioral problems include difficulty concentrating,memory loss,personality changes,distort reality and mental decline.About 20 to 30 percent of IBGC patients have at least one of these psychiatric disorders.Published results suggested that multiple genes related to the IBGC.SLC20A2 was the first gene reported to link with IBGC.The following studies suggested that the mutations in the SLC20A2 gene cause almost half of IBGC cases.The new findings reported that a small percentage of IBGC caused by the mutations of PDGFRB gene.Some cases of IBGC appear to be associated with changes in chromosome 2,7,9 and 14,but specific genes have not yet been identified.The SLC20A2 gene encodes a sodium-dependent phosphate transporter 2(Pit2)protein.This protein plays an important role in maintaining phosphate homeostasis within the body by transporting phosphate from the extracellular to the intracellular.The mutations of SLC20A2 gene lead to phosphate transport into cells ineffectively and further increased the level of phosphate in bloodstream.In the brain,the excess phosphate combines with the calcium to form the deposit of calciums.However,the further study about localization and function of SLC20A2 in the brain need to be done to explain why the calcification mainly occurs in the basal ganglia.The researchers found that Pit2 expressed widely in mice brain,including neuron,astrocytes and vascular endothelial cells,by biochemical analysis and immunostaining.These provided us with a novel view to understand that why we can observe the calcification in the brain,but they cannot explain how the calcification occurs first in the basal ganglia.As for research on the brain,a complex structure,biochemical analysis and immunostaining are relatively obscure methods,so if we want to better understand the mechanism of calcification,we firstly need to localize the Pit2 more meticulous.The homologous gene of SLC20A2 in Drosophila is CG42575(dPit).The Drosophila brain is a high complexity.Both of which make it possible to study the localization of Pit2 in Drosophila.The aim of this research is to generate the dPit:GFP Drosophila stock for localizing the dPit in Drosophila.In this study,we constructed three plasmids expressing sgRNA in vivo,and then integrated them into Drosophila genome with site-specific insertion.Meanwhile,another donor vector had been constructed for homology-directed repair.Finally,we got the dPit:GFP Drosophila stock with CRISPR/Cas9 system and embryo microinjection technique.The GFP had been inserted in the C-terminal of dPit.This stock lay a solid foundation for further researches about the location and function of dPit.Furthermore,this tool may provide us a new viewpoint to explain how the calcification mainly occurs in the basal ganglia.