Gene and Cell Therapy: Therapeutic Mechanisms and by Nancy Smyth Templeton

By Nancy Smyth Templeton

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Extra resources for Gene and Cell Therapy: Therapeutic Mechanisms and Strategies, Second Edition, Revised and Expanded

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Recombinants between the shuttle and backbone plasmids containing the whole vector genome are selected by appropriate antibiotic resistance markers. Once made, a new vector is plaque purified repeatedly in 293 cells (to remove any contaminating wild-type virus) and is then propagated to produce the required amounts of the vector. Under standard laboratory conditions, it is possible to produce up to 2 ‫ ן‬1013 viral particles from fifty 150-mm cell culture plates (about 109 293 cells). The recombinant Ad is easily purified from cell lysates on equilibrium cesium chloride density gradients.

In general, this has been achieved using 293 cells transfected with the appropriate E2 or E4 gene driven by an inducible promoter (Table 1). In a typical example, E4 deleted vectors were constructed in a cell line that expressed the E4 OFR6 behind an inducible metallothionein promoter (93). Cell lines of this type are more difficult to work with than 293 cells, and the efficiency of vector production is often lower. There is inconsistent data on whether additional genomic deletions result in a blunted immune response and whether this translates into longer persistence of transgene expression.

Fass D, Davey RA, Hamson CA, Kim PS, Cunningham JM, Berger JM. 0 angstrom resolution. Science 1997; 277:1662–1666. Barnett AL, Davey RA, Cunningham JM. Modular organization of the Friend murine leukemia virus envelope protein underlies the mechanisms of infection. Proc. Natl. Acad. Sci. USA 2001; 98:4113–4118. Lavillette D, Boson B, Russell S, Cosset F-L. Membrane fusion by murine leukemia viruses is activated in cis or in trans by interactions of the receptor-binding domain with a conserved disulfide loop at the carboxy-terminus of the surface glycoproteins.

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