Cancer-associated Gene Expression Vector And Recombinant Retrovirus Packaging

Study on the packaging of retrovirus vector combinant with c-myc Abstract Two expression plasmids,pLXSN-bcl-2 and pLXSN-c-myc were constructed by inserting the proto-oncogene c-myc and the apoptosis- inhibitory gene bcl-2 into the downstream of 5-LTR of replication- defective retrovirus vector pLXSN respectively. The two recombinant plasmids were confirmed to be correct by restrictive enzyme analysis. The the plasmid pLXSN-c-myc was transfected into the ecotropic packaging cell line W-2 by Lipofectin method. For 2 days, retrovirus were harvested from the supernatant of ??-2 and then used to infect the amphotropic packaging cell line PA3 17 in the presence of 8p.gImL polybrene.The PA317 cells infected were selected in 325 .tgImL Genegticin(G418)for 9 days. G418-resistant clones were isolated and their supernatant were evaluated primarily for the amphotropic retrovirus titer by infecting target cell line NIH3T3.The clone V producing higher titer retrovirus than others was selected and cultured. Pakaged retroviruses were harvested from the supernatant of clone V and concentrated through centrifuge at different speeds at 4 0C .They were evaluated again for the retrovirus titer.The results showed that the titer of retrovirus was 1.67 X 1O7CFU/mL, 50 times as high as that (3.5 X IO5CFU/mL) before the concentration.