Construction Of Eukaryotic Expression Plasmid Vector PEGFP-N1/EPO And Its Expression In Vitro

Objectivecloning EPO gene fragment of rat's kid ney, To construct rat eukaryotic expressionplasmid vector pEGFP-N1/EPO,culturing and identifing rat's MSCs cell, transfectingrecombina ted eukaryotic expression plasmid vector pEGFP-N1/EPO in rat's MSCs cell, todetect the EPO expression of the plasmid in rat's MSCs,which is the basis for further studyon the treatment of HIBD.Methods1. Construction and identifica tion of eukaryotic expression plasmid vector pEGFPN1/EPOWe gained EPO cDNA fragment of the rat's kid ney in sterile and RNA freeenvironment by RNAiso Reagent, amplified it by RT-PCR,cut gel and purified RT-PCRproduction; After liga ted EPO cDNA fragment with pMD19-T Simple Vector,wetransformed it in E.coli Competent Cells JM109,then cultured E.coli Competent CellsJM109 in LB agar pla te for one night.we chose white colony and amplified it in LB fluidmed ium, extracted plasmid vector by plasmid extraction kit and named plasmid vectorCTB865-T-1,CTB865-T-2,sent them to TaKaRa Biotechnology(Dalia n)Co. Ltd forsequencing and blast the outcome with EPO cDNA fragment of the rat's kid ney inGeneBa nk.The result was that the sequence of CTB865-T-2 was correct.we digestedCTB865-T-2 and pEGFP-N1vector by restriction endonuclease Hindâ…¢and Kpnâ… ,and theninserted the EPO cDNA fragment into pEGFP-N1 vector by TaKaRa DNA Liga tionKit,transformed it in E.coli Competent Cells JM109,cultured E.coli Competent Cells JM109in LB agar pla te for one night.we chose white colony and amplified it in LB fluidmed ium,extracted plasmid vector by plasmid extraction kit and named plasmid vectorCTB866-P-1.After that ,we identified plasmid vector pEGFP-N1/EPO by restrictionendonuclease digestion and nucleotide sequencing in sheng gong company in Sha ng hai. Separation, cultiva tion and identifica tion of rat's MSCs.At the same time ,we took out of the rat's integrity femur and tibia in sterileenvironment,separated the rat's prima ry MSCs by Percoll separating med ium,culture rat'sprima ry MSCs in DMEM/F12 med ium added with standard feta l bovine serum;d igested andcultured passage when cells fusion was about 80% in 0.25% pancreatic enzyme.After that,we assessed P5 MSCs by detecting CD44,CD45,CD90 expression in MSCs with FCM.3. Transfection of recombina nt plasmid pEGFP-N1/EPO into the rat's MSCs anddetection of the expression of EPO protein in vitroWe transformed accred ited CTB866-P-1 plasmid vector in E.coli Competent CellsJM109, then cultured E.coli Competent Cells JM109 in LB agar pla te for one night.we chosewhite colony and amplified it in LB fluid med ium,extracted endotoxin free plasmid byendotoxin free plasmid extraction and transfected it in the rat's P5 MSCs byLipofecta mineTM 2000,observed green fluorescence of MSCs in fluorescent lightmicroscope .The expression of EPO protein was detected by immunohistochemistrytechniq ues and RT-PCR in 48-72 hours.ResultResults1. The rat's EPO cDNA was gained successfully by RT-PCR.Recombina nt plasmidpEGFP-N1/EPO was verified by restriction endonuclease digestion and nucleotidesequencing.2. The rat's MSCs expressed CD44 and CD90,but don't express CD45.3. In MSCs cells ,EPO protein was expressed after transfection with the constructedrecombina nt plasmid by immunohistochemistry techniq ues and RT-PCR detection.ConclusionWe Separated and cultured rat's MSCs and constructed eukaryotic expression plasmidvector pEGFP-N1/EPO successfully.After transfection with the constructed recombina ntplasmid ,MSCs can express EPO in vitro.