Effects of sequences of prokaryotic origin on titer and transgene expression in retroviral vectors.

Transcriptionally targeted MLV-based ProCon vectors allow expression of the transduced gene in a promoter-specific manner by replacement of the viral U3 region with a heterologous promoter. In order to evaluate the effects of sequence elements present in ProCon vectors on transgene expression (enhanced green fluorescence protein, EGFP), a series of deletion constructs mimicking the situation in proviral DNA following promoter conversion, where expression of the EGFP gene is driven by three different constitutive promoters (MLV U3, mCMV, and hCMV) in the context of a 5'LTR, respectively, were generated and tested in transient transfection experiments. We discovered that modifications in the 3'LTR have only marginal effects on the EGFP expression and the sequence between the promoter and the transgene did not influence EGFP expression at all. On the other hand, EGFP expression was reduced by up to 17-fold in cells transfected with constructs containing SV40neo and/or pBR322ori sequences. To study this effect in transduced cells, we generated a series of retroviral vectors in which these elements were deleted in various combinations and found that an increase in EGFP expression and viral titer was also consistently obtained using vectors lacking these elements, although this was much smaller than that observed using the expression constructs. A vector containing the gene for puromycin resistance (pac) in place of the neomycin resistance gene (neo) was also tested, and found to result in improved vector titers and transgene expression. We conclude that, where possible, the inclusion of neo and ori sequences in retroviral vectors should be avoided, and that, if selection of infected cells is necessary, the pac, rather than neo gene should be used.