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Derivation
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They were derived from the human embryonic kidney line, 293 (see ATCC CRL-1573). Consistently higher transduction of target cells was achieved with ProPak-derived amphotropic vector than with PA317-packaged amphotropic vector.
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Comments
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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 derive the amphotropic packaging cell line ProPakA.6, the pCMVEa plasmid was introduced into 293 cells by co-transfection with the pHA58 plasmid conferring resistance to hygromycin B (250 mg/ml). Clones were subsequently transfected with gag-pol and vector plasmids. Next, the pCMV-gp construct was stably transfected into the 293-Env clones by cotransfection with the plasmid pSV2pac. The cells are puromycin-resistant (1 mg/ml). They secrete defective (non-infectious) murine leukemia virus (MLV) particles composed of gag-pol and env proteins. ProPak-A is a stable amphotropic packaging cell line in which the Gag-Pol and Env (packaging) functions are expressed separately from a heterologous (non-MLV) promoter, to maximally reduce homology between packaging and vector sequences A vector that consistently gives rise to replication-competent retrovirus (RCR) in PA317 cells never results in detectable RCR in ProPak-A-based producer cultures. 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.
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