Notes

1 The quality of the solution of DNA introduced into the egg is crucial to the success of the method. DNA fragments for microinjection should be purified as described in Chapter 11. The composition of the solvent in which the DNA is dissolved is extremely important. The presence of even small amounts of Mg2+ will kill an injected egg, as will an excessively high concentration of EDTA. It has been determined that the optimal buffer consists of 10 mM Tris-HCl, pH 7.4, containing EDTA to 0.1-0.25 mM (microinjection TE, or MITE; 7).

2 The DNA should be injected at a concentration of 1-5 mg/mL. Excessively high DNA concentrations can kill the injected egg (7), but gener ally there is no correlation between the number of DNA molecules introduced, and the structure or copy number of the resulting transgene.

3. The physical state of the injected DNA has little effect on the success of a transgenic experiment. Cosmid clones of up to 50 kb in length can be introduced with the same efficiency as smaller molecules (4). Linearized DNA fragments apparently are able to integrate at an efficiency fivefold greater than circular molecules (7). The structure of the DNA fragment ends created by different restriction endonucleases has no effect on the efficiency of generating transgenics, or on the organization of the resulting transgene (7,8).

4. Fertilized one-cell eggs should be maintained in M2 for as brief a period as possible (maximum 30 min to 1 h). Thus, eggs held in microdrop culture should be processed for injection in small, manageable batches. A skilled operator ought to be able to inject 20-40 eggs in 15 min.

5. When injecting the pronucleus, it is worth trying to avoid the sticky nucleolus. If this attaches to the injection pipet, it may be drawn out of the nucleus, thus killing the egg.

6. In the hands of a practiced investigator, 80% of eggs ought to survive the insult of microinjection.

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