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High-Titer ,Retroviral Vectors for Gene Delivery

y 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 then 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 virus is produced transiently as 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 (present in the virion particle), and the cDNA of the gene of interest, flanked by the LTRs, is integrated into the host DNA. Because the vector itself does not express viral proteins, once a target cell is infected, the LTR expression cassette is incapable of proceeding through another round of virus production.

Vector Description

Fig.2

The vectors pFB and pFB-Neo were designed to achieve the highest titers possible. They contain the bacterial origin of replication and the ampicillin resistance gene from pBR322 (Figure 2). It has been shown for the vector pMFG that a significant increase in viral titer is achieved by extending the 3 border of the Y site further into the gag coding sequence relative to other MMLV vectors.5 This extended packaging signal (Y+) has been incorporated into the pFB and pFB-Neo vectors. In addition, the MMLV splice acceptor (SA) sequence in pMFG has been inserted immediately downstream of the Y'"/>

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