ORF-mutated and promoter-deleted versions of the PRE are still functional. (a) The PRE fragments (oPRE, bPRE4*, LPRE) were inserted into the 3′UTR of self-inactivating gammaretroviral (SIN) vectors bearing identical transgene cassettes driven by an internal CMV promoter.26 SC-1 murine fibroblasts were transduced with the indicated vectors at a low multiplicity of infection (transduction efficiency <35%) to ensure the presence of a single vector copy in the majority of transduced cells. At 5 days after transduction analysis was performed by FACS and the MFI of the GFP-positive cells was determined. Vector titer is indicated on the right side. Error bars were calculated from three independent experiments. (b) Northern blot analysis of transduced SC-1 cells (<35% pos. cells) according to (a). Analysis was performed as in Figure 2a. A probe corresponding to the GFP cDNA was used (probe II in Figure 1b). RNA species are marked on the right. (c) Titer and expression analysis of gammaretroviral SIN vectors habouring different PRE fragments in murine hepatoblastoma cells (Hepa1.6). Mean values and s.d. of three independent experiments are shown. (d) Northern blot analysis of Hepa1.6 cells transduced with the indicated constructs. Preparation was performed as in Figure 2a. RNA species are named on the right. (e) Lentiviral vectors bearing the same PRE versions as in (a). Analysis according to (a) and mean values and s.d. of three independent experiments are shown. (f) Titer and expression analysis of LTR vectors containing different PRE fragments. Titer determination was performed on SC-1 cells and d2GFP expression was analyzed by FACS. Mean values and s.d. of three independent experiments are shown.