FIG. 1.
Retroviral vectors and virus surface protein expression vectors. Retroviral vector pLXSN (22) contains the neomycin phosphotransferase reporter gene (neor) under the control of the SV40 enhancer and early promoter. In order to generate retroviral vector pLXSP, which transduces the puromycin resistance gene (pac [i.e., puromycin acetyltransferase]), pLXSN was digested with NcoI, followed by ligation of the resulting 5,146-bp pLXSN fragment to the 848-bp fragment of NcoI-digested plasmid pBSpacΔP (8) containing the 3′-end of the SV40 early promoter together with the entire pac gene. Retroviral double-reporter vector pLZ12 (29) contains the neor gene under the control of the MoMLV long terminal repeat (LTR) promoter and enhancer and the β-galactosidase reporter gene attached to a nuclear location signal (nlslacZ) under the control of a truncated Rous sarcoma virus (RSV) LTR. VSV-G expression vector pcDNA3-G was constructed by inserting the 1.6-kb VSV-G cDNA XhoI-XhoI fragment of plasmid pSVGL1 (27) into the XhoI site of mammalian expression vector pcDNA3 (Invitrogen, Leek, The Netherlands). SV-F expression vector pcDNA3-F was constructed by ligation of the 1.7-kb SV-F cDNA EcoRI-XhoI fragment of pUC29-F (14) with EcoRI-XhoI-digested plasmid pcDNA3. Transient and stable transfections of all plasmids were carried out with 3 μg of DNA and 20 μl of Lipofectamine (Life Technologies, Eggenstein, Germany) per 5 × 105 packaging cells according the manufacturer’s instructions. 5′ LTR, Moloney murine sarcoma virus LTR; 3′ LTR-pA, MoMLV LTR; RSV, truncated RSV LTR; ψ+, extended packaging signal; CMV, human cytomegalovirus immediate-early promoter; SV40, SV40 early promoter and enhancer; SV-pA, SV40 polyadenylation site; BGH-pA, bovine growth hormone polyadenylation site. The figure is not drawn to scale.