Plasmid Constructs. The plasmid pBC.tTA (see Fig. 1) was constructed from pBC12/cytomegalovirus (CMV)/interleukin 2 (11) by replacement of the interleukin 2 sequences (bp 756–1439) with the tet transctivator gene from pUHD10–1 (12). To construct pMDtet.G (Fig. 1), the 1.6-kb EcoRI fragment from pSVGL (13) containing the VSV-G gene was cloned into the EcoRI cloning site in pMD.tet which is within exon 3 of the genomic human β-globin sequence. pMDtet was constructed with a 0.47-kb XhoI-BamHI fragment from pUHC 13–3 (12), which contains the tet operator and minimal human cytomegalovirus (HCMV) enhancer-promoter sequences, a 1.34-kb BamHI-XbaI fragment from pUCMdβs(R)S (14) that includes the genomic human β-globin sequences from the BamHI site in exon 2 through 690 bp in the 3′ untranslated region, and a 3.06-kb XbaI-XhoI fragment from pSL301 (Invitrogen).
To construct pMD.gagpol (see Fig. 1), PCR was performed with pCRIPenv− (15) using the following pairs of primers: 5′-CGGAATTCATGGGCCAGACTGTTACC-3′ and 5′-AGCAACTGGCGATAGTGG-3′, 5′-CGGAATTCTTAGGGGGCCTCGCGG-3′ and 5′-ACTACATGCTGAACCGGG-3′. The PCR products were digested with EcoRI and XhoI and with EcoRI and HindIII, respectively, to generate 0.94-kb EcoRI-XhoI and 0.94-kb HindIII-EcoRI fragments. These fragments were ligated with the 3.3-kb XhoI-HindIII fragment from pCRIPenv− and with pUC19, which had been linearized with EcoRI and calf intestinal phosphatase treated, to produce pUC19.gagpol. The 5.2-kb EcoRI fragment from pUC19.gagpol was cloned into the EcoRI cloning site in pMD to yield pMD.gagpol. pMD was constructed with the 3.1-kb EcoRI-BamHI fragment from pBC12/CMV/interleukin 2 that includes the pXF3 backbone and HCMV enhancer-promoter region and the previously described 1.34-kb BamHIXbaI fragment derived from pUCMdβs(R)S. The 3.1-kb EcoRI-BamHI and 1.34-kb BamHI-XbaI fragments were ligated after the EcoRI and XbaI overhangs were blunt-ended by Klenow treatment.
The plasmids pJ6Ωpuro and pJ6Ωbleo conferring resistance to puromycin and bleomycin (and zeocin), respectively, were kindly provided by J.Morgenstern (16). The plasmid pSV2neo confers resistance to G418 (17).
Retroviral Vectors. MFG.SnlsLacZ (see Fig. 1) was kindly provided by O.Danos (18). This vector is a derivative of MFG (19) in which mutations have been introduced at nucleotides 412 (A to T), 429 (T to A), and 631 (C to T) [nucleotide 625 of the Moloney murine leukemia virus (MuMLV) sequence]. These substitutions produce the sequence, ATGGGCCCGGGGTAG, thereby preventing expression of the N-terminal portion of gag that would otherwise be expressed by the vector. The ΔU3nlslLacZ retroviral vector was constructed by precise replacement of the U3 region in the 5′ long-terminal repeat of MFG.SnlsLacZ with the HCMV enhancer-promotor (bp −671 to −2) (20). For the construction of ΔU3nlsLacZ, a 701-bp fragment encoding the HCMV promoter was generated by PCR with the pMD plasmid as the template with the pair of primers, 5′-GGGCCCAAGCTTCCCATTGCATACGTTGTATC-3′ and 5′-GGACTGGCGCCGGTTCACTAAACGAGCTC-3′, creating a 5′ HindIII site and a 3′ KasI site. The PCR product was digested with HindIII and KasI to yield a 677-bp fragment. The 91-bp KasI-StyI was isolated from the 3′ long-terminal repeat of MFG (19). The 253-bp StyI-EagI and the 4994-bp EagI-ScaI fragments were isolated from MFG.SnlsLacZ, and the backbone for ΔU3nlsLacZ is a 2.65-kb HindIII-SmaI fragment from pUC18.
DNA Transfection. Stable transfection of 293 cells was performed by the calcium phosphate precipitation method (22) with 5 μg pBC.tTA, 5 μg pMDtet.G, and 1 μg pJ6Ωpuro. For all stable and transient transfections, plasmid DNA was prepared by double banding on CsCl density gradients (23). Cells (1.5×106) were plated on 60-mm dishes in 4 ml 293 growth media the night before transfection. Chloroquine (final concentration, 25 μM) and tetracycline (final concentration, 1 μg/ml) were added to the media 5 min before transfection. The media was changed 7 h posttransfection. The transfected cells were plated 48 h posttransfection by limiting dilution in media containing puromycin and tetracycline and independent clones were isolated.
293 G cells were always grown in 293 growth medium supplemented with tetracycline and puromycin (293G growth medium). Stable transfection of the 293G cells was performed by the calcium phosphate precipitation method with 10 μg pMD.gagpol linearized with ScaI and 2 μg pSV2neo. Cells (2×106) were plated on 60-mm dishes in 4 ml 293G media the night before transfection. Chloroquine (final concentration, 25 μM) was added to the media 5 min before transfection. The media was changed 7 h posttransfection. The transfected 293G cells were plated by limiting dilution 48 h posttransfection in 293G growth medium supplemented with G418 and independent clones were isolated.
293GPG cells were grown in 293G growth medium supplemented with G418 (293GPG medium). Stable transfection of the 293GPG cells with MFG.SnlsLacZ was performed by the calcium phosphate precipitation method with 12.5 μg MFG.-SnlsLacZ linearized with AseI and 2.5 μg pJ6Ωbleo linearized with AflIII. Cells (4×106) were plated on 60-mm dishes in 4 ml 293GPG media the night before transfection. The media was changed 9 h posttransfection. The transfected 293GPG cells were plated by limiting dilution 48 h posttransfection in 293GPG media supplemented with zeocin and independent clones were isolated.
Transient transfections with 293GPG cells were performed on 60-mm dishes where 4–5×106 cells were plated the night before in 4 ml 293 growth medium. Four micrograms of ΔU3nlsLacZ was diluted into 300 μl OptiMEM (GIBCO/ BRL) and incubated at room temperature for 30 min with 25 μl lipofectamine (GIBCO/BRL) diluted into 300 μl OptiMEM. The DNA-lipofectamine mixture was diluted into 2.4 ml OptiMEM and layered on top of the 293GPG cells, which had been rinsed 30 min before transfection and had media replaced with 2 ml OptiMEM. Seven to 8 h posttransfection, 2 ml 293 media was added, and the media was changed at 24 h with 2.5 ml 293 media. The supernatant was harvested at 72 h and viral titers determined as described below.
Analysis of VSV-G Expression in Transfected Cells. The pMDtet.G and pBC.tTA cotransfected clones were screened for inducible VSV-G expression by plating each clone in parallel into two 35-mm tissue culture dishes at 50% confluence. The following day one plate was rinsed twice with 2 ml 293G media without tetracycline and maintained in this media. At 48 h the postnuclear cellular lysates were prepared and the paired samples run on a 7.5% SDS/PAGE under reducing conditions. The gels were transferred onto nitrocellulose (0.45 mm; Schleicher & Schuell) with a semidry electroblotter (Owl Scientific, Woburn, MA). Western blot analysis was performed by using a murine monoclonal anti-VSV-G IgG (Sigma) at a dilution of 1:800 and a peroxidase-conjugated F(ab′)2 fragment donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories) was used at a dilution of 1:10,000. Detection by chemiluminescence was performed using commercially available reagents (Renaissance; New England Nuclear).
Assays For Reverse Transcriptase (RT) and β-Galactosidase Activity. 293G cells transfected with pMD.gagpol were screened for RT activity in the culture medium of subconfluent clones growing in 24-well culture dishes as described (24). Cells were stained for β-galactosidase activity as described (25).
Viral Titers, Virus Concentration, and Stability of Virus to Human Serum. To determine viral titers, NIH 3T3 cells were plated at 1×105 cells per well in 6-well culture dishes 16 h before infection and incubated for 24 h with serial dilutions of