Establishment of a cell line with stable expression of exopolyphosphatase (PPX)

来源 :中国神经科学学会第四次会员代表大会暨第七届全国学术会议(The 7th Biennial Meeting and the | 被引量 : 0次 | 上传用户:rongsiyouyu
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  Exopolyphosphatase (PPX) is one of the main enzymes that degrade and release orthophosphate (Pi) from the ends of polyphosphate.It is widely distributed in living organisms and the characteristics of this enzyme have been investigated in E.coli,yeast,sponge and mammals.The best studied exopolyphosphatase was scPPX1 found in Saccharomyces cerevisiae.We transfected C6 rat glioma cell line with either pcDNA3.1 plasmid (Control) orthe plasmid encoding ppxl (P4) by lipofectamine 2000.The transfected cells were selected with 1 mg/ml G418 and cloned.The isolated clones were screened for the expression of plasmids.We successfully established two stably transfected cell lines:"control" expressing only plasmid and "P4" expressing plasmid+scPPX1.Under ischemia,control cells began to detach from the culture dish at 4 hr ischernia and almost no cell remained attach at 16 hr.Most P4 cells retracted their processes and became round but remained attach to the culture dish at 8 hr ischemia.At 16 hr ischemia,P4 cells were dead as confirmed by trypan blue staining; but these dead P4 cells were able to retain their intact appearance and remain attach to the dish.
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