| Applications |
Virions produced from the transfected Psi-2 cells were used to infect the amphotropic packaging line PA317, and infected cells were selected in medium containing G418. The pLXSN16E7 vector contains the human papilloma virus (HPV) type 16 E7 gene under control of the Moloney murine leukemia virus (MoMuLV) promoter- enhancer sequences. The vector also contains a gene controlling resistance to neomycin transcribed from the SV40 promoter. This line produces the amphotropic retrovirus LXSN16E6 which encodes the HPV16 E7 open reading frame, and which can be used to stably infect many cell types. The resulting cells constitutively express the E6 protein of HPV16 which binds to and inactivates the retinoblastoma (Rb) gene. The virus can be used to immortalize human keratinocytes. |
| Derivation |
PA317 LXSN 16E7 is a packaging cell line developed by transfection of the retrovirus vector pLXSN16E7 into the Psi-2 ecotropic packaging cell line. Virions produced from the transfected Psi-2 cells were used to infect the amphotropic packaging line PA317, and infected cells were selected in medium containing G418. |
| Comments |
PA317 LXSN 16E7 is a packaging cell line developed by transfection of the retrovirus vector pLXSN16E7 into the Psi-2 ecotropic packaging cell line. Virions produced from the transfected Psi-2 cells were used to infect the amphotropic packaging line PA317, and infected cells were selected in medium containing G418. The pLXSN16E7 vector contains the human papilloma virus (HPV) type 16 E7 gene under control of the Moloney murine leukemia virus (MoMuLV) promoter- enhancer sequences. The vector also contains a gene controlling resistance to neomycin transcribed from the SV40 promoter. This line produces the amphotropic retrovirus LXSN16E6 which encodes the HPV16 E7 open reading frame, and which can be used to stably infect many cell types. The resulting cells constitutively express the E6 protein of HPV16 which binds to and inactivates the retinoblastoma (Rb) gene. The virus can be used to immortalize human keratinocytes. |
| 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: 1:6 to 1:12
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 |
Halbert CL, et al. The E7 gene of human papillomavirus type 16 is sufficient for immortalization of human epithelial cells. J. Virol. 65: 473-478, 1991. PubMed: 1845902
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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