1500 600 300 200 100 1500 600 300 200 100 Supplemental Figure 3. Kiem et al. a Copy Number in Foamy Transduced Lymphocytes 3 2 Copy Number 1 0 FV-SMPGW FV-UsI-SMPGW FV-HsI-SMPGW FV-SC46-IMPGW FV-UsI-SC46-IMPGW FV-UsIIUsIUR5-SMPGW FV-HsIIHsIHR5-SMPGW FV-HsIIHsIHR5-SC46-IMPGW Predicted Recombination products and PCR fragment sizes (bp) b c Analysis of recombination FV-SMPGW FV-HsI-SMPGW FV-UsI-SMPGW FV-SC46-IMPGW FV-UsI-SC46-IMPGW FV-UsIIUsIUR5-SMPGW FV-HsIIHsIHR5-SMPGW FV-HsIIHsIHR5-SC46-IMPGW Supplemental Figure 3. A. Copy number analysis of foamy transduced lymphocytes used for challenge assays and for analysis of siRNA expression by Northern blot. The copy number in CEM.NKR.R5 lymphocytes was determined by real-time PCR. All anti-HIV vectors were within a 3.3-fold range. B. Predicted recombination products of tandem Pol III promoters. PCR primers flanking the shRNA expression cassette amplify the indicated size DNA fragments in bp for the unmodified three Pol lII-driven shRNA cassette (middle), after recombination to generate a single Pol lII-driven cassette (top) or a dual Pol III-driven cassette (bottom). C.PCR analysis of recombination products in foamy transduced lymphocytes used for challenge assays and for analysis of siRNA expression by Northern blot. PCR was performed on DNA from transduced lymphocytes (top panel) and DNA from vector plasmid DNA (bottom panel) and the products analyzed by agarose gel electrophoresis and ethidium bromide staining. Efficient recombination was observed with the triple U6-driven Pol III promoter and partial recombination was observed with the triple H1 Pol III promoter.