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The vector gTR55 neoA was introduced into D3 embryonic stem (ES) cells by electroporation to disrupt the TNFR locus.

The cell line generated is deficient for the tumor necrosis factor receptor (TNFR) p55 gene.

The cell line was used to produce mutant mice with germ line transmission for the TNFR disruption.

Heterozygous mice were inbred to obtain mice homozygous for the disrupted gene.

The line should be grown on feeder layers of mitomycin C treated primary mouse embryonic fibroblasts or STO cells (see ATCC 56-X.2, MITC-STO cells).

The line should be grown on feeder layers of irradiated (3000 rads) or mitomycin C treated (0.01 mg/mL for 90 minutes) primary mouse embryonic fibroblasts or STO cells (see ATCC CRL-1503 or ATCC 56-X, irradiated STO cells).

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-0.03% (w/v) 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 with 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. To remove trypsin-EDTA solution, transfer cell suspension to centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes.
6. Discard supernatant and resuspend cells in fresh growth medium. Add appropriate aliquots of cell suspension to new culture vessels.
7. Place culture vessels in incubators at 37°C.

Subcultivation Ratio: 1:3
Medium Renewal: Every two to three 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.