Notes

1. The quality of the DNA is crucial to the survival of the eggs. DNA should be purified as described in Subheading 10.3., step 13. The composition of the solvent in which the DNA is dissolved is very important. The presence of minute amounts of Mg2+ will kill an injected egg as will an overly high concentration of EDTA. The optimal buffer is 10 mM Tris-HCl, pH 7.4, containing 0.1-0.25 mM EDTA (Microinjection TE or MITE; 10).

2. There is no correlation between the concentration of DNA and the resultant copy number of the transgene. However, excessively high concentrations of DNA are toxic to the egg (10).

3. An estimated 1-2 pL of DNA solution enter the egg pronucleus in each microinjection. Most investigators, use concentrations of 1-5 mg/mL and are thus injecting about 500 copies of the DNA fragment depending on the size of the fragment (10). For different fragments, the DNA concentration combining the optimum efficiency of integration and egg survival will vary. One can make up different dilutions of the DNA (1, 2, and 5 mg/mL) and rotate between these dilutions during a microinjection session.

4. The physical state of the DNA has little bearing on the success of the experiment. Cosmid clones of 50 kb can be introduced as easily as DNA of smaller sizes (20) Linearized DNA integrates with a fivefold greater efficiency than circularized DNA (10). The structure of the DNA ends created by different restriction enzymes has no effect on the efficiency of integration or the organization of the resulting transgene (10).

5. Avoid the sticky nucleolus on microinjecting. If this attaches to the injection pipet, it may be drawn out of the egg, thus killing it.

6. A technique has been developed in which DNA complexed with polysine is microinjected into the egg cytoplasm rather than the pronuclei (thus affording a much easier and larger target), 12.5% of pups born from zygotes injected in this manner were transgenic compared with 21.7% for pronuclear injection. No transgenic pups were born from microinjection DNA alone into the cytoplasm (21).

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