Experimental Studies Of Using Retrovirus-mediated Fcy:: Fur Suicide Gene Combined With 5-FC In Treatment Of Glioma

The purpose is to study anticancer effects of fusion suicide gene Fcy::Fur/5-fluorocytosine therapies to glioma cells C6. A pair of PCR primer for Fcy::Fur genes was designed according to the multiclone site of retroviral expression vector pLXSN and the sequence of Fcy::Fur, in which EcoR I and BamH I sites were introduced at the two 5'ends of the primers respectively. Then Fcy::Fur genes were amplified from the pORF5-Fcy::Fur using PCR . After DNA sequencing and analysis , Fcy::Fur genes were further inserted into pLXSN with sense open reading frame. The recombinant expression plasmids, which had been confirmed by enzymes digestion, were transfected into packing cells PT67. Supernatant of the cloned packing cells were harvested and tested for the presence of the recombinant retrovivus. Cells C6 were infected with recombinant retrovivus and selected by neomycin(G418) ,and the C6 cell lines expressing Fcy::Fur stably were then obtained. The results of PCR and RT-PCR demonstrated that Fcy::Fur gene had been integrated into the chromosome of resistant C6. After administration of 5-FC, the changes of these cells were observed through electron microscopy and the cytotoxicity efficacy was evaluated by MTT methods,...