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《Acta Academiae Medicinae Militaris Tertiae》 2007-01
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Construction of mouse PPARδ adenovirus vector and its expression in INS-1 cells

JIANG You-zhao~1,ZHANG Ling~2,ZHOU Yan~3,CHEN Bing~1(~(1)Department of Endocrinology,~(2)Outpatient Department,~(3)Breast Diseases Center,Southwest Hospital,Third Military Medical University,Chongqing 400038,China)  
Objective To construct the recombinant adenovirus vector containing mouse PPARδ,and infect the INS-1 cells.Methods The pCDNA3.0-mPPARδ plasmid was digested with KpnⅠ and EcoRⅤ to obtain full-length coding sequence of mPPARδ,then the mPPARδ cDNA was ligated to the pAdtrack-CMV vector.The plasmid of pAdTrack-mPPARδ was linearized with PmeⅠ,and the fragment was reclaimed and transformed into BJ5183 which contains pAdeasy.After being screened,the extracted plasmid of positive bacteria linearized with PacⅠ was transfected into HEK293 cells with lipofectamine~(TM) 2000 and was identified by the green fluorescence protein(GFP).The harvested virus was amplified in HEK293 cells and infected INS-1 cells.After infection,the expression of PPARδ was proved by GFP expression and Western blotting.Results The recombinant adenovirus vector containing mouse PPARδ was constructed successfully and the titer was 1.5×10~(10) pfu/ml.The PPARδ expression in INS-1 cells infected by the virus was much higher than the controls.Conclusion The constructed recombinant adenovirus vector containing mouse PPARδ provides a potent tool to investigate its biological function in pancreatic islets and other tissues.
【CateGory Index】: R346
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