| Applications |
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). They secrete defective (non-infectious) murine leukemia virus (MLV) particles composed of gag-pol and env proteins. 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. |
| Comments |
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. |
| Subculturing |
Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.
- Remove and discard culture medium.
- Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
- Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
- Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
- Add appropriate aliquots of the cell suspension to new culture vessels.
- Incubate cultures at 37°C.
Subcultivation Ratio: 1:4 to 1:10
Medium Renewal: Every 2 to 3 days
Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 10 in Culture of Animal Cells, a Manual of Basic Technique by R. Ian Freshney, 3rd edition, published by Alan R. Liss, N.Y., 1994. |
| References |
Rigg RJ, et al. Method for obtaining retroviral packaging cell lines producing high transducing efficiency retroviral supernatant. US Patent 6,017,761 dated Jan 25 2000
Rigg RJ, et al. A novel human amphotropic packaging cell line: high titer, complement resistance, and improved safety. Virology 218: 290-295, 1996. PubMed: 8615038
Forestell SP, et al. Novel retroviral packaging cell lines: complementary tropisms and improved vector production for efficient gene transfer. Gene Ther. 4: 600-610, 1997. PubMed: 9231077
Hay, R. J., Caputo, J. L., and Macy, M. L., Eds. (1992), ATCC Quality Control Methods for Cell Lines. 2nd edition, Published by ATCC.
Caputo, J. L., Biosafety procedures in cell culture. J. Tissue Culture Methods 11:223-227, 1988.
Fleming, D.O., Richardson, J. H., Tulis, J.J. and Vesley, D., (1995) Laboratory Safety: Principles and Practice. Second edition, ASM press, Washington, DC.
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