Mice homozygous null for the caveolin-1 gene, Cav-1 (-/-), and their wild-type littermates, Cav-1 (+/+), were generated by targeted disruption of the caveolin-1 gene. A construct was introduced into WW6 embryonic stem (ES) cells by electroporation to disrupt the Cav-1 locus. Mouse embryonic fibroblasts (MEFs) were obtained from day 13.5 littermate mouse embryos and immortalized using the 3T3 protocol.xa0RefRazani B, et al. Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities. J. Biol. Chem. 276: 38121-38138, 2001. PubMed: 11457855

The 3T3 MEFs KO cell line (ATCCxa0CRL-2753) is homozygous for a disruption of the caveolin-1 gene Cav-1 (-/-) while the 3T3 MEFs WT cell line (ATCCxa0CRL-2752) is Cav-1 (+/+). Analysis of cultured fibroblasts from Cav-1 null embryos reveals a loss of caveolin-2 protein expression; defects in the endocytosis of a known caveolar ligand, (fluorescein isothiocyanate-albumin); and a hyperproliferative phenotype.xa0

These phenotypic changes are reversed by recombinant expression of the caveolin-1 cDNA.xa0RefRazani B, et al. Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities. J. Biol. Chem. 276: 38121-38138, 2001. PubMed: 11457855

A culture deposited with the ATCC in September of 2002 was found to be contaminated with mycoplasma. Progeny were cured by a 21-day treatment with BM Cyclin. The cells were assayed for mycoplasma, by the Hoechst stain, PCR and the standard culture test, after a six-week period following treatment. All tests were negative.

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-053mM EDTA solution to remove all traces of serum which 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.

Subcultivation Ratio: 1:5 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.

Razani B, et al. Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities. J. Biol. Chem. 276: 38121-38138, 2001. PubMed: 11457855

Sotgia F, et al. Intracellular retention of glycosylphosphatidyl inositol-linked proteins in caveolin-deficient cells. Mol. Cell. Biol. 22: 3905-3926, 2002. PubMed: 11997523

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.