| Derivation |
The ANJOU 65 cell line is a derivative of the 293T (293tsA1609neo) cell line.
293T is a highly transfectable derivative of the 293 cell line into which the temperature sensitive gene for SV40 T-antigen was inserted.
293T cells were cloned and the clones tested with the pBND and pZAP vectors to obtain a line capable of producing high titers of infectious retrovirus, 293T/17 (see ATCC CRL-11268).
These cells constitutively express the simian virus 40 (SV40) large T antigen, and clone 17 was selected specifically for its high transfectability.
293T/17 cells were cotransfected with the pCRIPenv- and the pCRIPgag-2 vectors to obtain the ANJOU 65 cell line.
ANJOU 65 cells were cotransfected with the pCRIPgag-2 and pGPT2E vectors to obtain the BOSC 23 ecotropic envelope-expression packaging cell line.
ANJOU 65 cells were also cotransfected with the pCRIPAMgag vector along with a plasmid expressing the gpt resistance gene to obtain the Bing (see ATCC CRL-11554) amphotropic envelope-expression packaging cell line. |
| Comments |
The ANJOU 65 cell line is a derivative of the 293T (293tsA1609neo) cell line.
293T is a highly transfectable derivative of the 293 cell line into which the temperature sensitive gene for SV40 T-antigen was inserted.
293T cells were cloned and the clones tested with the pBND and pZAP vectors to obtain a line capable of producing high titers of infectious retrovirus, 293T/17 (see ATCC CRL-11268).
These cells constitutively express the simian virus 40 (SV40) large T antigen, and clone 17 was selected specifically for its high transfectability.
293T/17 cells were cotransfected with the pCRIPenv- and the pCRIPgag-2 vectors to obtain the ANJOU 65 cell line.
ANJOU 65 cells were cotransfected with the pCRIPgag-2 and pGPT2E vectors to obtain the BOSC 23 ecotropic envelope-expression packaging cell line.
ANJOU 65 cells were also cotransfected with the pCRIPAMgag vector along with a plasmid expressing the gpt resistance gene to obtain the Bing (see ATCC CRL-11554) amphotropic envelope-expression packaging cell line. |
| Subculturing |
Do not let monolayer get too heavy or cells will slough off.
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 1:8
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 |
Pensiero M, et al. Retroviral vectors produced by producer cell lines resistant to lysis by human serum. US Patent 5,952,225 dated Sep 14 1999
Pensiero M, et al. Retroviral vectors produced by producer cell lines resistant to lysis by human serum. US Patent 6,329,199 dated Dec 11 2001
Pear WS, et al. Production of High-Titer Helper-Free Retroviruses by Transient Transfection. Proc. Natl. Acad. Sci. USA 90: 8392-8396, 1993. PubMed: 7690960
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.
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.
|
