To establish U87 cell line which steadily expresses epidermal growth factor receptor-variantⅢ.

EGFRvIII cDNA (2 832 bp) was cloned into XbaⅠ and NotⅠ sites of vector pCDH-CMV-MCS-EF1-PURO, and His6-tag was added in the primer for sequencing. The lentivirus shuttle plasmid containing pCMV-dR8.9, envelope plasmid pVSV-G and vector plasmid pCDH-CMV-EGFRvIII-EF1-puro were transfected to 293T cells by calcium phosphate system. After virus was filtrated, and concentrated and packaged, the viral titer was detected by dilution counting method. The prepared U87 cells were used to establish cell line with stable expression by puromycin screening and 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.

The reconstructed vector was digested by XbaⅠ and NotⅠ and underwent agarose gel electrophoresis, as the result EGFRvIII cDNA (2 832 bp) matched the expected size. Western blotting found that EGFRvIII protein and His6 protein at 145 kD were expressed in 6 strains of U87-EGFRvⅢ cells. Flow cytometry found that 2 strains of U87-EGFRvIII cell (U87-vIII-2, U87-vIII-4) expressed EGFRvIII protein (93.1% and 96.9% respectively) on surface.

We have successfully designed and constructed U87 cell lines with stable expression of EGFRvIII, which provide usefull foundation for further EGFRvIII targeting studies around glioma.

Epidermal growth factor receptor variant Ⅲ; Glioma; U87