| Comments |
Introduction of retroviral vectors by infection or transfection results in the production of retrovirus virions capable of infecting cells from many species (with the exception of mice).
Selection against loss of the DNAs conferring the packaging functions can be performed by growing the cells in medium containing dialyzed fetal bovine serum and 100 nM amethopterin for 5 days followed by cultivation in medium containing HAT and untreated fetal bovine serum for an additional 5 days.After selection, the cells should be maintained in medium containing HT for 2 days to avoid toxic effects due to residual amethopterin.
A derivative of this line is available (PG13/LN c8, see ATCC CRL-10685) that produces a neomycin resistance transducing vector.
Although virus production has not been observed, there is a possibility that the cells will produce a virus similar to GaLV (a moderate risk oncogenic virus) and Biosafety Level 2 or higher precautions should be taken when using this cell line. |
| Subculturing |
Protocol: Remove medium, add fresh 0.25% Trypsin, 0.53 mM EDTA solution and allow the flask to sit at room temperature (or 37C) until the cells detach (2 to 3 minutes). Add fresh medium, aspirate and dispense into new flasks. Subcultivation Ratio: A subcultivation ratio of 1:8 is recommended Medium Renewal: Every 2 to 3 days |
| References |
Miller AD, et al. Construction and properties of retrovirus packaging cells based on gibbon ape leukemia virus. J. Virol. 65: 2220-2224, 1991. PubMed: 1850008
Miller AD, Rosman GJ. Improved retroviral vectors for gene transfer and expression. BioTechniques 7: 980-990, 1989. PubMed: 2631796
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.
Biosafety in Microbiological and Biomedical Laboratories, 5th ed. HHS. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. Washington DC: U.S. Government Printing Office; 2007. The entire text is available online.
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