Construction and preliminary application of retroviral vector which carries EGFP gene.

Objective To construct the recombinant retroviral vector which carries enhanced green fluorescent protein(GFP) gene, analyze the level of GFP expressing in vitro and in vivo and observe the biological character of cell expressed EGFP gene. Methods EGFP gene was amplified by PCR and directionally inserted into the multiple cloning site of retroviral vector pLXSN. Positive clone was identified by enzyme digesting and PCR. Transfected replication defective retroviral vector was packaged by PA317 cells into SW038-C2 cells. The expression of EGFP gene in tumor cells and the biological character changing of tumor cells was observed in vitro and in vivo by fluorescence microscope and FACS. Results Successfully constructed the recombinant retroviral vector pLXSN-EGFP and was packaged by PA317. The recombinant vector can transfect the glioblastoma mutiforme cells of human SW038-C2 and express normally. The growth of transfected cells and tumor nods were not affected. The cells expressing high level EGFP gene became long and thin. Assaying by FACS, about 98% of these cells expressed GFP. The intensity of the fluorescence was not reduced after 6 months cultured in vitro. The shape of cells expressing low level EGFP gene is normal and 30% of them expressed GFP. Conclusion The EGFP gene could express normally in vitro and in vivo and didn't influence the growth of tumor cells. But the morphology of tumor cells expressed GFP gene was changed. Using GFP as the report gene, transfection rate of the retroviral vector is lower than expected.