Factors Affecting the Success of Nuclear Transplantation Based Transgenesis

1. Egg quality is a major factor that contributes to the level of postgastrula development, which is obtained from sperm nuclear transplantation. To obtain good postgastrula development, eggs must be generally healthy. In particular, they should have even pigmentation and should be firm enough to hold their shape well after dejellying. In addition, it is important that they do not become acti vated before they are injected with nuclei. When egg quality is poor, a fraction of the embryos will show morphogenetic defects, resulting in incomplete blastopore closure during gastrulation. This problem is often compounded by expressing genes at high levels during the gastrula stages. Therefore, embryos expressing genes from the CMV promoter are more likely to show nonspecific gastrulation defects than embryos expressing genes from strong promoters that are turned on after gastrulation.

2. Transplantations should be performed and eggs incubated after transplantation at temperatures no higher than 22°C. We have found that transplantation and early incubation of activated eggs at elevated temperatures (24-25°C) lowers the frequency with which plasmids are expressed in batches of nuclear transplantation-derived embryos. Embryos in these batches also frequently express plasmids in only one-quarter to one-half of the expected cells. We believe that acceleration of the first cell cycle, which occurs at 24-25°C, may give these embryos inadequate opportunity to integrate introduced plasmids prior to first cleavage, thus resulting in more chimeric and nonexpressing embryos.

3. It may be important to note that although this technique is very efficient and workable, it involves several steps; all of which are critical for its success. Therefore, we suggest that anyone trying to learn the technique does so in steps, rather than all at once. For example, one should first learn to isolate sperm nuclei and transplant them into eggs. Once this can be done successfully, resulting in normal development, then one can determine whether sperm nuclei, swollen in extracts, gives normal development. If swelling of sperm in extract has no adverse effects on the level of development obtained after transplantations, one can add plasmid and enzyme to the reaction, thus reconstituting the whole transgenesis procedure.

4. Dilution drastically changes the pH of HEPES, making it impossible to pH the stock directly.

5. For each sperm nuclei prep, it is most convenient to make about 30 mL of 2X NPB form stock solutions. Then use this 2X stock to make all subsequent solutions (i.e., 1X NPB + protease inhibitors, 1X NPB + 3% BSA, and so forth).

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