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High-Titer ,Retroviral Vectors for Gene Delivery

>+. The SA splices to the natural splice donor (SD) located 58 bp downstream of the 5 LTR. Thus, expression from the 5 LTR in the vector (as well as in the ultimate integrated provirus) yields unspliced and spliced transcripts. The unspliced transcript contains the Y+ site and is packaged into virus particles in packaging cells. The spliced transcript has a relatively short, efficiently translated mRNA leader and allows high-level gene expression from the provirus. This splice-site configuration has also been incorporated into the pFB vectors.

The vectors pFB and pFB-Neo differ only by the presence of the IRES/neomycin-resistance gene cassette in the vector pFB-Neo. The open reading frame (ORF) for the neomycin-resistance (neo) gene is positioned downstream of the MCS, using the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES). Expression of neo from the second ORF in a dicistronic message is advantageous in that G418-selection (i.e., selection for neo-expression) ensures that the transcript containing the gene of interest is efficiently expressed. Furthermore, the potential loss of titer due to inclusion of an exogenous promoter between the LTRs6,7 is avoided by coexpressing both proteins from a single transcript.

In light of published reports showing that a significant increase in titer is achieved by minimizing the amount of extraneous sequence between the LTRs,3,7 we also constructed the vector pFB, which contains only the sequences required for virus production, in addition to an MCS. Because of limitations in insert size due to packaging constraints, a related advantage of pFB is that elimination of extraneous sequences allows the insertion of cDNA
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