PA317 LXSN 16E6E7 (ATCC® CRL-2203)

Organism: Mus musculus, mouse  /  Tissue: embryo  /  Disease: Papilloma

Permits and Restrictions

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Organism Mus musculus, mouse
Tissue embryo
Product Format frozen
Morphology fibroblast
Culture Properties adherent, adherent
Biosafety Level 2 [Cells contain human papilloma viral DNA sequences]
Disease Papilloma
Age embryo
Strain NIH/Swiss
Applications
This cell line produces the amphotropic retrovirus LXSN16E6E7 which encodes the HPV16 E6 and E7 open reading frames, and which can be used to stably infect and immortalize many cell types.
Storage Conditions liquid nitrogen vapor phase
Derivation
PA317 LXSN 16E6E7 is a packaging cell line developed by transfection of the retrovirus vector pLXSN16E6E7 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
The pLXSN16E6E7 vector contains the human papilloma virus (HPV) type 16 E6 and E7 genes 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.
Complete Growth Medium The base medium for this cell line is ATCC-formulated Dulbecco's Modified Eagle's Medium, Catalog No. 30-2002. To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 10%.
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.

  1. Remove and discard culture medium.
  2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.
  3. 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.
  4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
  5. Add appropriate aliquots of the cell suspension to new culture vessels.
  6. Incubate cultures at 37°C

Subculture Ratio: 1:6 to 1:12
Medium Renewal: 2 to 3 times a week.
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.

Cryopreservation
Freeze medium: Complete growth medium supplemented with 5% (v/v) DMSO
Storage temperature: liquid nitrogen vapor phase
Culture Conditions
Temperature: 37°C
Name of Depositor DA Galloway
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

Basic Documentation
Restrictions

Note: The user of the PA317 LXSN 16E6E7 cell line agrees to indemnify and hold harmless the United States, the ATCC, Denise A. Galloway and Fred Hutchinson Cancer Research Center, Seattle, Washington from any claims, costs, damages, or expenses resulting from any injury (including death), damage or loss that may arise from the use of the cell line either directly (including use for diagnostic purposes) or in the preparation of a product. The user assumes all risks and responsibilities in connection with the receipt, handling, storage and use of the material.

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