( that can be used with any MMLV-based r...)Safe Production of High-Titer Retrovirus

that can be used with any MMLV-based retroviral vector to produce viral supernatants with titers that are consistently greater than or equal to10⁷ cfu/ml in transient triple-transfection experiments. The CMV-based^### vectors include a gag-pol-expressing vector that is cotransfected with the retroviral expression vector together with one of four envelope (env)-expressing vectors. Choosing which vector to use depends on what range of cell types is desired for transduction. Because all of the cis and trans elements required to produce infectious virus are separated onto three plasmids, with minimal or no sequence overlap between the plasmids, the pVPack system is much safer than the majority of stable producer cell lines or vector-based systems, where there is a large degree of homology between the packaging vector(s) and the retroviral expression vector. In these latter systems, there is a relatively high probability of production of replication-competent retrovirus (RCR) due to homologous recombination between the vectors.¹

Although the relative speed and simplicity of the transient high-titer virus production methods used here are attractive for most applications, the gag-pol and env open reading frames (ORFs) are all followed by an internal ribosome entry site (IRES) linked to a downstream drug-resistance gene so that these vectors may also be used to establish stable producer lines. The antibiotic-resistance genes used in the gag-pol and env vectors are different from each other, and from those used in most of the more popular retroviral vectors; hence, any env vector may be used with the gag-pol vector and with a wide range of antibiotic-resistant retroviral vectors to produce triple-stable viral producer lines. The position of the antibiotic-resistant gene as the second ORF in a dicistronic expression cassette, as opposed to its expression from a second cassette on the s
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