Model Construction Of GPS2-knockout Mouse And Its Phenotype Analysis

G-protein suppressor2was firstly found while searching for the suppressors ofRas activation. GPS2is widely expressed in various tissues and species and involvedin inflammation, obesity, cholestrol metabolism, erythroid differention and brainmorphogenesis in vitro study. However, the physiological function of GPS2in vivoremains unknown and the corresponding antibody applied for recognizingendogenous mouse GPS2is also rare.We first generate GPS2antibody. GPS2cDNA were amplified from mouse testisand subcloned into T-vector, and the recombinant T-GPS2plasmid was produced.Then GPS2cDNA fragment from T-GPS2plasmid was then inserted into pET28band generate pET28b-GPS2recombinant vector which expresses GPS2protein fusedwith His tag. Then the recombinant GPS2protein was produced for immunizingrabbits and mouse. A rabbit polyclonal antibody against GPS2and13strains mousemonoclonal cell clones were obtained. The reactive activity of these proteins againstmouse endogenous GPS2was also confirmed.To furthur explore the function of GPS2in vivo, we knockout the GPS2gene inC57BL/6mouse using ZFN (zinc finger nuclease) technology. Analyzing theoffspring of GPS2+/-mice suggest that none GPS2-/-mice was produced, indicating aembryo lethality of homozygous GPS2-knockout mice. The phenotype of GPS2-/-embryo include pale yolk sack with vague vascular, Reichert’s membrane with bloodclot and much thinner placenta.The embryo bodies grow smaller than the wild typewith hemorrhage and vague vascular. Also, in some embryos, the somites was notinverted in time. Some embryos were digested. Cardiac pericardium bulge andallantois not merge with chorion also occured in some embryos.To investigate the mechanism from lethal phenotypes of GPS2knockout mouse,the expression profile of GPS2from C57BL/6wild type mouse was investigated.GPS2is widely distributed in all of the tested tissues, and it is highly expressed intestis and lung. Through the whole embryo TUNEL experiments, we found theapoptosis increase. Using whole embryo immunofluorescence assay, we found theapoptosis also increase in gene knockout embryo and the capillaries of embryo didn’tform a good vessel network. Realtime PCR analysis suggest that the hematopoietic transcription factor gata1and lmo2were downregulated and the vessel developmentrelated gene c-met was upregulated in GPS2-knockout embryo. These results suggestthat GPS2may play an important role in embryo development.