Safe Production of High-Titer Retrovirus ( litates infection of the target cell by...)

Safe Production of High-Titer Retrovirus

litates infection of the target cell by direct interaction with cell type-specific receptors; thus the host range of the virus is dictated not by the DNA vector but by the choice of the env gene used to construct the packaging cell.

The packaging cell line is transfected with the vector DNA and, at this point, either stable viral producer cell lines may be selected (providing the vector has an appropriate selectable marker) or mRNAs that are transiently transcribed from the vector are encapsidated and bud off into the cell supernatant. These supernatants are collected and used to infect target cells. Upon infection of the target cell, the viral RNA molecule is reverse transcribed by RT (which is present in the virion particle), and the cDNA of the gene of interestflanked by the LTRsis integrated into the host DNA. Because the vector itself carries none of the viral proteins, once a target cell is infected, the LTR expression cassette is incapable of proceeding through another round of virus production.

Due to recent advances in transfection technology high-titer viral supernatants can be produced following transient cotransfection of the viral vector together with expression vectors encoding the gag, pol, and env genes (Figure 1).^3,4 This obviates the need for the production and maintenance of stable packaging cell lines.

Vector Description

Fig.2

The general structure of the packaging vectors is diagrammed in Figure 2. The vectors were designed with four aims in mind:

High Titer: MMLV gag-pol fusion protein and each of the envelope proteins are expressed at sufficiently high levels to allow the production of viral supernatants with titers greater than or equal to 10⁷ cfu/ml following transient transfection.
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( litates infection of the target cell by...)Safe Production of High-Titer Retrovirus

Vector Description