( ted samples were selected with ...)High-Titer ,Retroviral Vectors for Gene Delivery

ted samples were selected with 1 mg/ml of G418 for 10 days, then the colonies were stained with methylene blue and counted. The titer, as determined by G418 resistance, was identical to that determined by FACS (1 x 10⁷ cfu/ml) (data not shown).

Fig.4

To assess expression levels from the pFB vectors, the GFP coding sequence was inserted into the MMLV vector pLXSN,¹⁰ and viral titers were determined for the derivative pLXSN-GFP and pFB-Neo-GFP. NIH3T3 cells were subsequently infected with an equivalent number of virus particles for both vectors, and expression levels were qualitatively measured by fluorescence intensity in the population as a whole by FACS (Figure 4, Panel A and Panel B) and in individual cells by fluorescence microscopy (Figure 4, Panel C and Panel D). As the results in Figure 4 indicate, the peak fluorescence intensity is several-fold higher for cells infected with pFB-Neo-GFP and is also significantly higher on a cell-to-cell basis for this vector.

Conclusions

Using the vectors pFB and pFB-Neo for high-titer virus production will, in many cases, obviate the need for scale-up production and tedious concentration steps when large numbers of virus particles are required (e.g., production of cDNA expression libraries). In addition, the relatively high expression levels achieved with these vectors will facilitate library screening and enhance general-purpose gene expression in infected cells. Because the vectors may be used with any MMLV-based packaging line, virus can be produced that infects virtually any cell type. The relatively small amount of sequence between the LTRs allows for expression of sequence of up to 8 kb without a significant loss of titer. The small size also facilitat
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