Microinjection of RNA and of the lineage tracer fluorescein-lysine-dextran uses the same technique, except that it is essential to bake injection needles used for RNA to destroy RNase activity. As stated above, there are several different kinds of injection apparatus, and our description is therefore rather general.
1. Transfer embryos to be injected to 4% Ficoll in 75% NAM.
2. Attach the injection needle to the micromanipulator and injection system. Dispense about 2 |L of water onto a small piece of parafilm, and suck it up into the pipet. This rinses the pipet and allows one to calibrate it.
3. Calibrate the injector/needle combination by injecting into a dish of sterile mineral oil. Calculate the volume injected by the formula V = 4/3nr3. A volume of about 10 nL can be safely injected into a fertilized egg.
4. Expel water, and load the injection sample.
5. Inject embryos. This is most easily done by supporting the embryo with a pair of forceps held in the left hand (this is for a right-hander again) and using the right hand to control the micromanipulator. Injections can be made into any part of the egg, but for uniform distribution of the injected material, it is best to aim for the equatorial region, where the pigmented animal hemisphere meets the paler vegetal hemisphere. A few hundred embryos can be injected in an hour.
6. Culture embryos in 4% Ficoll in 75% NAM until stage 8.
Was this article helpful?