Isolation of Primordial Germ Cells

1. Mice: ICR mice are an outbred strain that can be used to generate large numbers of PGCs. These can be obtained from a number of suppliers, including Taconic Farms (Germantown, NY, 1-800-822-6642) or Harlan Sprague-Dawley (Indianapolis, IN, 1-317-894-7521). Noon on the day of plug is 0.5 d postcoitum (dpc). See Note 1 for discussion of different mouse strains.

2. Dissecting tools: Fine scissors and forceps (watchmaker's #5) for the dissections. Diamond pen for cutting glass pipets. These can be ordered through Fisher (Atlanta, GA, 1-800-766-7000) or VWR (Marietta, GA, 1-800-932-5000). More specialized dissection equipment is available from Roboz Surgical Instruments (Rockville, MD, 1-800-424-2984) or Fine Science Tools (Foster City, CA, 1800-521-2109).

3. Tissue-culture equipment, dishes, pipets, and so on, can all be obtained through Fisher or VWR. Manufacturers include Corning, Falcon, and Nunc.

4. Tissue-culture reagents can be purchased from Grand Island Biological Company (Gibco, Grand Island, NY, 1-800-828-6686). Trypsinization solution is 0.25% trypsin, 1 mM EDTA in Hank's balanced salts (cat. no. 25200-056). Dulbecco's phosphate-buffered saline (PBS) is 0.2 g/L KCl, 0.2 g/L KH2PO4, 8 g/L NaCl, 1.15 g/L Na2HPO4, and 2.16 g/L Na2HPO47H2O. (cat. no. 14190). Medium is Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 4.5 g/L glucose (cat. no. 11965-084). For the culture of PGCs, this medium is further supplemented with 0.01 mM nonessential amino acids (cat. no. 11140-019), 2 mM glutamine (cat. no. 25030-016), 50 pg/mL gentamycin (cat. no. 15750-011), 0.1 mM 2-mercaptoethanol (cat. no. 21985-023), and 15% fetal bovine serum (ES cell tested, from either Summit Biotechnology, Fort Collins, CO, 1800-933-0909 or HyClone Laboratories, Logan, UT, 1-800-492-5663). For the routine culture of fibroblast feeder cells, the DMEM with glucose is supplemented with 10% fetal bovine serum, 2 mM glutamine, and 50 pg/mL penicillin-streptomycin (Gibco cat. no. 15070-014). Collagenase/dispase can be purchased from Sigma (1-800-325-3010) (cat. no. C3180), made up to 1% (1 g/100 mL) in either DMEM or PBS, and stored in aliquots at -20°C. This is a 10X concentration. Dimethyl sulfoxide (DMSO) for freezing cell lines is available from Fisher Scientific (cat. no. BP231-1). Bovine serum albumin (BSA) for dissections is available from Sigma (cat. no. A 9647).

5. Feeders for primary cultures are Sl4-m220 cells (from David Williams, Indiana University School of Medicine), whereas those for the secondary culture are generated from mouse embryos (see Notes 2 and 3 for more details about feeder cells). Sl4-m220 is a cell line isolated from homozygous Steel mutant embryos and stably transfected with the membrane-associated form of SCF. Growth factors necessary for the generation of EG cell lines are LIF (ESGRO™, Gibco cat. no. 13275-011), bFGF (also called FGF2) (Gibco cat. no. 13256-029) and SCF (also known as Steel factor, mast cell growth factor, and c-kit ligand) (R&D Systems, Minneapolis, MN, 1-800-343-7475, cat. no. 455-MC or Preprotech, Rocky Hill, NJ 1-800-436-9910 and London, 0171 603 8288, cat. no. 250-03). See Note 3.

6. Feeder layers are mitotically inactivated by y-irradiation or by mitomycin treatment. Mitomycin can be obtained from Sigma (cat. no. M0503), and a 1 mg/mL stock is stored frozen in PBS in a dark container. Mitomycin C is very toxic, and care should be used when handling (gloves, mask, and so on) (see Note 4).

7. Dissecting microscope with low-power objectives (10x-80x).

8. A mouth-controlled pipet should be used for most of the manipulations. They can be purchased from Fisher (cat. no. 258616).

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