Establishment Of Glioma Cell Line With Stable Expression Of EGFRvⅢ

Objective Epidermal growth factor receptor variant III is a most common mutant of epidermal growth factor receptor,which was formed in the absence of the exocyte2-7 exogenous cell in EGFR.The mutant present to tumour cells,rather than normal tissue,so it’s a good target for tumour targeted therapy.It is a tumor specific antigen with high expression rate of glioma and other tumors,which associated with many malignant phenotypes of tumors.It is high-profile target for tumour targeted therapy.Glioma is the most common malignant brain tumor in adults,although there are many traditional treatments,but they have failed to achieve satisfactory curative effect.Recently,targeted therapy regarded as a new idea for glioma immunotherapy.We have successfully designed and constructed U87 cell lines with stable expression of EGFRvⅢ,which provide usefull tool in further EGFRvⅢ targeting studies around glioma.Establishment of U87 cell line with stable expression of epidermal growth factor receptor-variant III,providing usefull tool in further EGFRvⅢ targeting studies about glioma.Methods EGFRvⅢ cDNA(2832bp)was cloned into XbaI and NotI sites of vector pCDH-CMV-MCS-EF1-PURO,his-tag was added in the primer.The lentivirus was obtained by using pCMV-dR8.9,pVSV-G and pCDH-CMV-EGFRvⅢ-EF1-puro to co-transfect 293 T cells.After filtration and concentration of packaged virus,the viral titer was detected by using the dilution counting method.The prepared EGFRvⅢ lentivirus infect U87 cells.The puromycin screening and method of limited dilution were adopted to stabilize the transfected cell line.Western blot and Flow cytometry were used to detected the expression of EGFRvⅢ and His6.Results The reconstructed vector was digested by XbaI and NotI before agarose gel electrophoresis,EGFRvⅢ cDNA(2832bp)matched the expected size.Western blotting detected EGFRvⅢ protein and his6 protein at 145 kD in 6 strains of U87-EGFRvⅢ cells.Flow cytometry found that 2 strains of U87-EGFRvⅢ cell(U87-vIII-2,U87-vIII-4)express EGFRvⅢ protein(93.1% and 96.9% respectively).Conclusions We have successfully designed and constructed U87 cell lines with stable expression of EGFRvⅢ,which provide usefull tool in further EGFRvⅢ targeting studies around glioma.