OBJECTIVETo establish the BGC-823/WTX-EGFP gastric cancer cell line with stable expression of Wilms tumor gene on the X chromosome (MTX) for functional analysis of WTX gene.

METHODSThe full-length WTX cDNA was amplified from human embryonic kidney 293FT cells and cloned into the pEGFP-N1 vector containing the reporter gene of green fluorescence protein. The recombinant pEGFP-WTX expression vector, after digestion by restriction enzyme to identify the size of target gene fragment, was transfected into 293FT cells and the expression of fluorescent reporter gene was observed under fluorescence microscope. pEGFP-WTX vector was transfected into human gastric cancer BGC-823 cell line to establish BGC-823/WTX-EGFP cell line stably expressing WTX. Quantitative RT-PCR and immunocytochemical staining were used to detect the expression of WTX in both BGC-823/WTX-EGFP and control BGC-823 cells.

RESULTSThe recombinant pEGFP-WTX plasmid was successfully constructed and verified by PCR and sequencing. The mRNA and protein expressions of MTX were significantly increased in BGC-823/WTX-EGFP cells as compared with those in the control cells.

CONCLUSIONThe full-length WTX expression vector (pEGFP-WTX) and BGC-823/WTX-EGFP gastric cancer cell line have been successfully established to facilitate further functional study of WTX gene.