( Produce high...)Safe Production of High-Titer Retrovirus

Produce high-titer retroviral supernatants with safe packaging vectors

Peter Vaillancourt Coleen Miller
Stratagene

We describe a set of vectors that can be used with any MMLV-based retroviral vector to produce viral supernatants that have titers that are consistently greater than or equal to 10⁷ cfu/ml following transient transfection. Because all of the cis and trans elements required to produce infectious virus are separated onto three plasmids, the probability of producing undesired replication-competent virus is very low. The range of target cells to be infected is determined by which one of four available envelope (env)-expressing vectors is cotransfected with the viral vector. The gag-pol and env open reading frames are all followed by an IRES linked to a downstream drug-resistance gene so that these vectors may be used to produce stable producer lines.

In recent years, tissue culture systems have been developed for the production of high-titer recombinant retrovirus that are capable of infecting a virtually limitless range of cell types. These systems have had a tremendous impact on fields for which very efficient gene delivery is essential.¹ With Moloney Murine Leukemia Virus (MMLV)-based vectors, transduction efficiencies of greater than 90% are achievable for most mitotic cell types, and the copy number per cell can be easily controlled by varying the multiplicity of infection. These are clear advantages over most standard transfection methods; typically, only a small population of transfected cells is capable of the uptake and stable integration of vector DNA, and the copy number is unpredictable and often prohibitively high for many applications, such as cDNA library expression screening.²

Stratagenes pVPack retroviral system comprises a set of five vectors
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