Figure 1

MMTV-based vector system. (A) Schematic representation of the MMTV provirus and the four-plasmid expression system used for generating MMTV-based vectors. For the MMTV provirus the regions encoding structural, enzymatic and accessory proteins are shown. The splice donor (SD) and acceptor (SA) sites with their nucleotide coordinates, the long terminal repeat (LTR), the Rem responsive element (RmRE) and the packaging signal are also indicated. In the transfer vector, pRRpCeGFPWPRE25, the truncated gag gene is followed by the Rev responsive element (RRE) from HIV-1 and the internal CMV promoter that drives the expression of enhanced green fluorescent protein (EGFP). Woodchuck hepatitis virus posttranscriptional element (WPRE) upstream of the 3′ LTR is also indicated. In the packaging construct pCMgpRRE17, the restriction sites used for the construction of this chimera, SD, SA, RRE and the beta globin polyadenylation signal (βg pA) are shown. The Rev-encoding plasmid (pRSV-Rev) and the VSV-G envelope protein-encoding plasmid (pHCMV-G) have been previously described [14, 22]. (B) and (C) Infectious titers of VSV-G-pseudotyped vectors (transduction units [TU]/ ml) determined by flow cytometric analysis on transduced HeLa cells three days post transduction using un-concentrated (B) or concentrated (C) supernatant from transfected 293 T cells. The mean values from three infection replicates are shown, together with the standard deviations (SD). (D) Time-course analysis of EGFP positivity after single exposure of HeLa cells to the MMTV- or HIV-1-based vectors. The cells were analysed by FACS at various time points (days 3-48) after transduction. Transductions were performed in triplicate. All values are means ± SD. The nucleotide coordinates are based on the prototypic MMTV strain BR6 (GenBank Acc. no.15122).