Production of high-titer retroviral vectors and detection of replication-competent retroviruses

Retroviral infection or calcium phosphate-mediated DNA transfection has been used for the generation of retrovirus producing cell lines through the introduction of vector DNA into the chromosomes of packaging cells. To compare the ability of the methods for DNA delivery to produce high-titer virus, we generated stable retroviral vector producing cell lines by the transfection or infection of a LN-based vector DNA into PA317 cells and assayed individual clones for production of virus. Of eight randomly chosen G418-resistant clones generated by transfection, only one clone produced the vector at up to >10(7) cfu/ml. Two of the five clones generated by infection yielded higher-titer viruses in the absence of helper virus - up to 5 X 10(7) more than the transfected clones, The titer of retroviral vectors can be increased by multiple rounds of infection through long-term incubation of amphotropic virus producing cells with ecotropic virus vectors. Such amplification of vector copy number resulted in increase in vector titer of up to 20-fold, For the experiments presented here, we have used an improved vector/packaging system designed for minimizing the possibilities for the generation of an replication-competent retrovirus (RCR). However, the potential of RCR generation was detected in the culture medium harvested from the highest-titer virus producing PA317 clonal cells generated by amplification of the vector through the modified cocultivation technique, although the generation of RCR is very infrequent in the system.