Packaging cell lines

Retroviral packaging cells are designed to supply the ┬┐raws-acting proteins required for replication and packaging of the engineered retroviral vector construct. Engineering these packaging cell lines has been accomplished by stable transfection of gag, pol and env into suitable cell lines. Constitutive high levels of helper expression are ensured by selection for drug resistance genes which are linked to the gag, pol and env expression cassettes. Interestingly, the gag and pol proteins from murine retroviruses can be used with a number of different env proteins

(including nonmurine env proteins) to create 'pseudotyped' vectors with different host ranges. A series of increasingly safer generations of retroviral packaging cell lines have been engineered to prevent the generation of replication competent retroviruses (RCR, see below) (Figure 3, Table 1).

Evolution of retroviral packaging cell lines

The first generation The earliest retroviral packaging cells were constructed by stable transfection of a complete provirus from which only the i|) (packaging) site had been removed. These cell lines were prone to replication competent retrovirus (RCR) generation since only one recombination event was needed to restore wild-type virus. Examples of these first-generation packaging cell lines include i|>-2, i|j-AM, and PA 12.

The second generation The second generation of retroviral packaging cells replaced the 3' I.TR with the SV40e poly-A signal to reduce the probability of generating RCR. In addition, the helper genome 5' LTR was mutated to destroy the integrative ability of this helper genome. Thus, even if a defective helper genome happened to get packaged, it would not be

Wild-type/ "helper1

virus

Wild-type/ "helper1

virus

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