Staining With XGal

Incubate reconstructed embryos in X-gal fix for 5 min at room temperature, wash three times in X-gal wash, then transfer them into staining solution until the color develops. The cumulus cells from ZIN40 mice stain with X-gal, whereas the oocytes within cumulus masses are negative (see Fig. 4A). Oocytes from D2 mice do not stain with X-gal (see Fig. 4B). Figure 4C shows a blastocyst developing from a D2 oocyte in which the nucleus was replaced with one from a ZIN40 cumulus cell. The staining in all nuclei of the blastocyst indicates reactivation of the ZlN40 genome. Blastocysts recovered from normally fertilized ZlN40 females also stain in this way (not shown). The blasto-cyst shown in Fig. 4C would have been a suitable candidate for derivation of ES cells expressing the transgenic marker from the donor nucleus.

4. Notes

1. It is very important that the osmolarity of all manipulation and culture media is within the recommended range. Too high a value may result in suboptimal development, whereas too low a value will cause lysis of oocytes during injection. It may be helpful to use a high osmolarity (~280 mosmoles) for CZB during the manipulation stages and transfer to a lower osmolarity (~265 mosmoles) after activation. The osmolarity can be increased by cautious addition of sucrose.

2. The mice from which the oocytes are harvested ideally are 4 to 12 wk old. Oocytes from older females are more likely to be low in number and have a tendency to abnormality after superovulation. lt is less critical that the cumulus donor mice fall within this age range.

3. The quality of hormones used for superovulation may affect the quality of oocytes. Do not store frozen aliquots for more than a few months and thaw immediately before injection.

Fig. 4. Expression of lacZ reporter gene confirms nuclear contribution in nuclear transfer embryos derived from transgenic somatic cell nuclei. Cumulus cells derived from ZIN40 transgenic mice display characteristic blue X-gal stained nuclei (A), whereas D2 oocytes do not stain (B). Nuclear transfer blastocyst, derived from direct injection of ZIN40 cumulus nuclei into D2 oocytes that were subsequently enucleated, displayed blue X-gal stained nuclei characteristic of the donor cumulus cell (C). Scale bar represents 100 pm.

Fig. 4. Expression of lacZ reporter gene confirms nuclear contribution in nuclear transfer embryos derived from transgenic somatic cell nuclei. Cumulus cells derived from ZIN40 transgenic mice display characteristic blue X-gal stained nuclei (A), whereas D2 oocytes do not stain (B). Nuclear transfer blastocyst, derived from direct injection of ZIN40 cumulus nuclei into D2 oocytes that were subsequently enucleated, displayed blue X-gal stained nuclei characteristic of the donor cumulus cell (C). Scale bar represents 100 pm.

4. Hydrofluoric acid is extremely toxic and will destroy any glass container. Store safely in a plastic container and wear protective clothing during handling. Ensure that acid, water and isopropanol solutions contain no impurities.

5. Mercury is toxic and must be handled carefully. Wear gloves at all times and dispense in an area where spills can be easily contained. Mercury spillage kits are available from Scientific Laboratory Supplies.

6. The shape of the injection and enucleation pipets is critical. They must be long enough to allow control of flow within the fine part of the barrel but not so long that their flexibility prevents controlled penetration of the zona. The correct shape of pipets can be decided only by trial and error.

7. Creating the ideal pipet for injection or enucleation requires some skill and practice. Fusing the filament to the appropriate point near the tip of the needle without causing thickening or distortion of its walls may present some difficulty at first.

8. Acid etching of injection and enucleation pipets will help guard against blockages during manipulation. The thinness of the walls at the tips of the pipets that is achieved by acid etching allows the penetration of the zona and oolema with minimal intensity and velocity of the piezo pulse, which reduces lysis of the oocyte and damage to the donor nucleus.

9. It is important to align holding and manipulation pipets correctly before performing either enucleation or injection. Failure to do so may increase the risk of lysis.

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