Study On Gcm2 Gene Knock-out Inducing The Hypoparathyroidism In Adult Mice

BackgroundGcm(glial cells missing)was firstly found in drosophila neural precursor cells and its differentiation into glioblast would be obstacle if Gcm gene was deleted in Drosophila.In mammals,two homologues,Gcm1 and Gcm2 have been found.Gcm1 is mainly expressed in placental labyrinth cells and involved in syncytiotrophoblast cell differentiation and chorionic formation.Gcm2 expresses in parathyroid cells as a transcription factor especially.It plays a vital role in the differentiation of patathyriod in early embryo and the survival of parathyriod in the late embryonic.Mouse knocked out of Gcm2 gene in early embryos can be observed in parathyroid absence.Mutation of human Gcm2 gene can lead to congenital familial isolated hypoparathyroidism.Studies have shown that Gcm2 is still expressed in adult parathyroid cells,suggesting that the Gcm2 gene may play an important role in mature parathyroid cells in the development of parathyroid embryos.However,the study of the Gcm2 gene is currently focused on its early stage of embryonic development,a nd the traditional gene knockout technique knocks out the Gcm2 gene early in the embryo and can not study its function in adult parathyroid cells for further research.Cre/Lox P tamoxifen induced conditional gene knock-out technology is a commonly used gene knock-out method in recent years.This technique fuses the ligand binding region of the estrogen receptor with Cre recombinase to form a chimeric recombinase.The chimeric recombinase can not bind directly to the Loxp site,and only give exogenous tamoxifen canit have recombinant function.So the knoc-kout can be achieved in time control.In this study,we used the Cre/Lox P knockout system to create an inducible Gcm2 conditional knock-out mouse line for the first time and achieved the control of Gcm2 knockout in time.Mice were injected intraperitoneally with tamoxifen to induce knock-out of the Gcm2 gene after maturation in mice.Detection of calcium,phosphorus metabolism and proliferation of parathyroid cells in mice after Gcm2 gene knockout to further explore the function of Gcm2 gene in adult mouse parathyroid cells.It also provided experimental basis for further study of new gene therapy targets of parathyroid dysfunction.Methods1.Screening the genotype of Gcm2E2fl/fl F1 passage mice by generation of Gcm2E2fl/+ of F0 passage mice.Then hybridizing them with Cre-ER transgenic mice to get genotypes of Gcm2E2fl/+Cre-ER and Gcm2E2fl/+ F2 passage mice.The two genotypes of F2 passage mice mated with each other,or backcrossd with F1 passage mice.Eventu ally we got genotype of Gcm2E2fl/flCre-ER inducible conditional knock-out mice;2.6-week-old mice were intraperitoneal injected with tamoxifen continuous for 5 days to induce Gcm2 knock-out;3.Identification of knock-out mouse genotype by PCR;4.The expression of Gcm2 protein in parathyroid gland was detected by Western blotting;5.The levels of serum calcium?phosphorus?parathyroid hormone(PTH)and 1,25(OH)2D were measured by calcium?phosphorus assay kit and ELISA kit respectively;6.Ki-67 immunohistochemical staining to observe the proliferation of parathyroid cells;7.Detection of Gcm2,PTH and Ca SR m RNA expression in parathyroid and thymus tissues by Real-time PCR;8.Detection of bone metabolism by Micro CT.Results1.Inducible conditional gene knockout mice with genotype Gcm2E2 fl / flCre-ER were obtained by three generations of breeding;2.Gcm2 gene of parathyroid was confirmed to be knocked out by PCR after tamoxifen administration;3.Compared with the wild type and solvent control group,the expression of Gcm2 protein in Gcm2 knockout group was significantly decreased(P <0.05);4.The serum concentration of Gcm2 knockout group was significantly increased(P <0.05),serum calcium and PTH concentrations were significantly lower(P <0.05),blo od 1,25(OH)2D concentration was no significant change.Parathyroid size and cell morphology were no significant changes;5.Ki-67 immunohistochemical staining showed that Gcm2 knockout group increased the proliferation of parathyroid cells(P <0.05);6.The expression of PTH and Ca SR m RNA in the parathyroid was significantly down-regulated(P <0.05).The expression of PTH m RNA was significantly up-regulated(P <0.05),while the expression of Ca SR m RNA was not significantly different;7.The bone volume(BV),relative bone volume fraction(BV/TV),bone trabeculae number(Tb.N),thickness(Tb.Th)and bone mineral density(BMD)were significantly increased in the ROI region of the femur(P <0.05);Conclution1.Tamoxifen 100 mg/kg continuous injection for 5 days induced adult mouse(Gcm2E2fl/flCre-ER)Gcm2 gene knockout and Gcm2 gene knockout development of Hypoparathyroidism;2.The m RNA expression of Ca SR and PTH in parathyriod was significantly decreased after Gcm2 knock-out,and the expression of PTH m RNA in thymus cells was significantly increased;3.Parathyroid cells may have compensatory proliferation after adult mouse Gcm2 gene knockout;4.Bone volume(BV),relative bone volume fraction(BV/TV),bone mineral density(BMD),number(Tb.N)and thicknes(Tb.Th)of trabecular were significantly increased in adult mice after Gcm2 knockout.