The ProPak packaging cell lines produce either murine leukemia virus (MLV) xenotropic particles (ProPak-X cells; ATCC CRL-12007) or amphotropic particles (ProPak-A cells; ATCC CRL-12006 and ATCC CRL-12479). They were derived from the human embryonic kidney line, 293 (see ATCC CRL-1573). To construct the ProPak-X and the ProPak-A-52 cell lines, the ATG in the splice donor/splice acceptor of pCMV plasmid was mutated to ACG. The CMV promoter was excised (EcoRI/XhoI, blunt-ended), and replaced with the MoMLV LTR (Asp 718/HindIII, blunt-ended) from plasmid pVH2. The beta-galactosidase gene was replaced by the gag-pol ORF (NotI fragment) to generate pMoMLVgp. pMoMLVgp was co-transfected with pHA58 into 293 cells by calcium phosphate co-precipitation and hygromycin B-resistant cells were selected. Clones were screened for the level of Gag secretion and one clone secreting high levels of Gag was selected (designated ProGag); this clone yielded high viral titers in transient transfection. The expression plasmid containing the murine xenotropic env gene, pCI-Ex, was co-transfected with pSV2pac into the ProGag cell line by calcium phosphate precipitation and puromycin-resistant cells were selected. The resulting cells were screened for Env expression and clones designated ProPak-X, expressing high levels of Env were screened for ability to produce transducing vector. Clone 36 designated ProPak-X.36 was deposited as CRL-12007. ProPak-based producer cells were demonstrated to be free of replication-competent retrovirus (RCR) by stringent testing. Consistently higher transduction of target cells was achieved with ProPak-derived amphotropic vector than with PA317-packaged amphotropic vector. The highest transduction of human hematopoietic progenitor cells was achieved with vector supernatant generated from a coculture of the ProPak-X and ProPak-A cell lines. |
