( + . The SA splices to the natural ...)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 LTRs^6,7 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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