1. DMEM+: For all procedures, we use Dulbecco's Modified Eagle's Medium (DMEM— Flow Labs [Finland], powder, cat. no. 430-1600) supplemented with the following:
a. 0.1 mM nonessential amino acids (100X stock, Gibco, Grand Island, NY, cat. no. 320-1140AG; see Note 1).
b. 1 mM sodium pyruvate (100X stock, Gibco cat. no. 320-1360).
c. 100 mM P-mercaptoethanol (100X stock stored as aliquots at -20°C, Sigma, St. Louis, MO, cat. no. 600564AG).
d. 2 mM L-glutamine (100X stock, stored as aliquots at -20°C, Gibco cat. no. 320-5030AG).
f. Penicillin and streptomycin (final concentration 50 pg/mL each, Gibco cat. no. 600-564AG).
g. Leukemia inhibitory factor (different sources, for example, Gibco) 1000 U/mL. The supplemented DMEM used for propagation of ES cells is referred to as DMEM+ (see Notes 1-3).
2. 0.1% Gelatin: 1 g (w/v) gelatin is 0.1% gelatin (Sigma or BDH) made up in 1 L water, autoclaved and stored at 4°C (see Note 4).
3. 2X ES cell-freezing medium: 2X ES cell-freezing medium should be made up fresh each time it is to be used, and should comprise freshly prepared 60% DMEM+, 20% FCS, and 20% DMSO (Sigma, cat. no. D-5879).
4. Phosphate-buffered saline (PBS): For all tissue-culture work, we use PBS without calcium and magnesium. This is made from 10 g NaCl, 0.25 g KCl, 1.5 g Na2HPO4, 0.25 g KH2PO4, pH 7.2. The solution is autoclaved and stored at 4°C.
5. Trypsin (0.1%): Dissolve 0.5 g trypsin powder (Gibco, cat. no. 0153-61-1) in 500 mL saline/EDTA solution. Adjust the pH to 7.6, sterilize through a 0.22-pm filter, and store at -20°C. This constitutes a 5% stock, which needs to be diluted to 0.1% on defrosting.
6. Saline/EDTA solution: 1L of saline/EDTA solution comprises 0.2 g EDTA, 8.0 g NaCl, 0.2 g KCl, 1.15 g Na2HPO4, 0.2 g KH2PO4, 0.01 g phenol red, 0.2 g glucose, pH 7.2. The solution should be sterilized through a 0.22-pm filter, and stored at room temperature.
7. Tissue-culture-treated plasticware: We routinely use NUNC, Corning, and Falcon plasticware.
8. Humidified incubator: This is maintained at 37°C and 5% CO2.
9. Electroporation apparatus: Use apparatus, such as a Bio-Rad GenePulser, and appropriate cuvets (Bio-Rad, Hercules, CA, cat. no. 165-2090).
10. Selection reagents: There are drugs, such as G418 (Gibco, cat. no. 11811-031), gancyclovir (Syntex, cat. no. 00865516), puromycin (Sigma, cat. no. P8833), and 6-thioguanine (Sigma, cat. no. A4660) or HAT (Gibco, cat. no. 31062-037) for HPRT-negative or positive selection, and mitomycin C (Sigma, cat. no. M-0563).
11. Cre recombinase-expression vectors: Cre recombinase-expressing vectors are described in detail in a number of publications (34-36).
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