1. ICR mice have been used for many PGC studies. However, depending on the objective of the experiment, and especially if EG cell lines are to be generated, it may be desirable to use inbred mouse strains, such as C57BL/6 or 129/Sv, to be assured of a uniform genetic background. Additionally, both of these have been used to generate EG cell lines that will contribute to the germ line of chimeras (15-17). Inbred mouse lines, such as C57BL/6 and 129/Sv, are available from Taconic Farms, Harlan Sprague-Dawley (Subheading 2.1., item 1), and The Jackson Laboratories (Bar Harbor, MA, 1-207-288-3371).

2. Primary mouse embryonic fibroblasts (mefs) can be generated from embryos between 12.5 and 14.5 d pc, but with 13.5 d pc embryos giving the highest yield of cells. Embryos are dissected free of their extraembryonic membranes and the heads, limbs, and visceral organs removed. The carcasses are then passed five times through an 18-gage needle (approx 5 embryos/syringe) in approx 3 mL of PBS, and the resulting cell mixture is plated into a 150-mm tissue-culture dish with fibroblast culture medium. After 3 d, the cultures are trypsinized and replated at a dilution of 1:4, and this secondary culture can then be mitotically inactivated or cultured further to generate more cells. These feeders can be frozen in 10% DMSO/ 20% fetal bovine serum in DMEM and stored indefinitely in liquid nitrogen. See Subheading 2.1., item 4 for a description of tissue-culture reagents and suppliers.

3. It is absolutely essential to use feeder cells that are producing SCF on their cell surface. If Sl4m220 cells are not available, STO fibroblasts (American Type Culture Collection [ATTC]) can be used (14). STO cells are available from the ATTC (1-800-638-6597, cat. no. ATCC CRL 1503). Primary mefs do not make sufficient amounts of SCF to allow the PGCs to survive and divide (15). It also may be important to use species-specific growth factors. For example, rat SCF can be used to promote the survival mouse PGCs, but human SCF will not affect the growth of mouse PGCs. This may be a limiting factor for the generation of EG cell lines in species other than mouse, but this information is primarily anecdotal.

4. To inactivate feeder cells mitotically, use >5000 rad of y-irradiation. Alternatively, mitomycin C can be added to the medium at 10 |g/mL and incubated in the 37°C incubator for 2-3 h. The cells are then washed very well with PBS (five changes of PBS), and trypsinized and plated as usual. Both methods are equivalent, but irradiation requires an expensive and specialized machine, so most investigators use mitomycin C. It is important to take safety precautions with mitomycin C (wear gloves and a mask), since it is very toxic.

5. Paraformaldehyde must be dissolved in PBS and heated to 65°C before it will go into solution. Care should be used when handling paraformaldehyde in order to avoid contact with fumes as well as the liquid or powder form.

6. For many manipulations of the starting cell cultures, a finely pulled Pasteur pipet is used with mouth control. It generally takes some practice both to use the mouth pipet as well as to pull the Pasteur pipets. The long, thin end of the pipet is broken off the wider part by using a diamond pen to score the glass. Then, the center of the long, thin part is softened in the small flame of a microburner or alcohol burner (a traditional Bunsen burner produces too violent a flame for this purpose), removed quickly from the flame, and pulled in one quick motion. Score the thin glass between the two ends with a diamond pen and break the glass, generating blunt ends. If necessary, the ends can be flame polished to make a smoother opening. Glass capillary tubes can also be used for this. The internal diameter of the pipet will vary with the task, so it is often a good idea to pull many pipets of various sizes (from 30 to 200 |im internal diameter). It is also important that these pipets be kept sterile so that the resulting cultures do not become contaminated. This is most easily accomplished by making the pipets shortly before use and keeping the pipets stored in a 150-mm tissue-culture dish during the dissection.

7. Most of the dissections are done with bright-field illumination, but at the end of the tissue dissociation, the use of dark-field illumination makes it easiest to see single cells. This is especially important in order to retrieve single cells from dissociated genital ridges.

8. PCR for the Zfy gene detects the presence of a Y chromosome. However, it should be noted that an EG cell line that is negative for this PCR product may actually be an XO cell line, not a normal XX female cell line. It would be necessary to perform karyotype analysis to determine the difference between an XO and an XX cell line accurately.

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