① Overview
This time, the eggs of goldfish and zebrafish which can remove the egg membrane are used. Because these eggs are floating in the egg cytoplasm, they can be placed in the cytoplasm of the embryo at any time during early development when the solution is injected into the egg. However, eggs that cannot remove egg membranes require new techniques, such as inserting needle tips from the hilum. In eggs with a single yolk bulb and a thin cytoplasm around it, the solution may have to be injected only into the thin cytoplasm. In this case, delicate injections must be made.
The solution to be injected into the egg does not include calcium ions. This is because the influx of calcium may cause contraction of the cytoplasm. 0. 2M KCL, PBSs, etc. are used as the types of solutions used.
② FITC-dextran, biotin-dextran solutions (cell-labeled)
Labeled cells are transplanted and injected with 5% FITC-dextran for external follow-up. After transplantation, 5% biotin-dextran-fixable solutions are injected into FITC-dextran solutions for histologic follow-up of the cells. When the transplanted embryo is fixed in Bouin's solution, the lysine on the dextran is cross-linked with the intracellular protein. Because the biotin is immobilized in the cells, an avitin that reacts with it can be used to colorize and locate the transplanted cells. Since these solutions have high viscosity, it is better to use a needle having a wide constriction portion.
③ mRNA solutions (PGCs-fluorescent)
mRNA solutions are injected to impart fluorescent to PGCs. Since this solution is transparent, it is difficult to confirm the amount entered during injection. Therefore, when injecting a GFP-fluorescent mRNA, it is preferable to mix a little rhodamine into the solution. For dsRed fluorescence, mix FITC, but less should be used because of the strong fluorescence.
④ Solution injection procedure
1. Centrifuge the test tube containing the solution to be injected and drop the dust down.
2. Take a solution of about 3 μl with a pipette man p20 fitted with a gel-loader tip. This should be taken from the top of the solution.
3. Under a stereomicroscope, place the tip of a narrowed glass needle on the side of the Petri dish and split the tip slightly.
4. Insert the gel loader tip behind this glass needle and place the solution. At this time, when the remaining liquid is placed at the tip of the guerroader tip, pull the tip of the tip to the front side of the liquid contained in the needle, and then put it in.
5. Attach the needle containing the liquid to the end of the injector holder. At this time, do not loosen the plastic part at the end of the holder too much. If it is loosened too much, the liquid entering the needle may go too far to the tip when this part is tightened to the metal part. Tighten the plastic part at the tip properly.
6. Gently turn the knob on the injector body to move the solution to the tip.
7. Attach a holder to the manipulator.
8. Loosen the screw supporting the manipulator a little, rotate the manipulator, and apply the tip of the glass needle to the Petri dish containing the egg.
9. Focus the stereomicroscope on the egg.
10. Rotate the manipulator's xyz-axis knob to attach the tip of the needle to the egg.
11. Check if the solution comes out of the tip of the needle. If there is a large amount, turn the injector knob slightly counterclockwise to reduce the amount. If not, turn the knob clockwise so that it flows out a little.
12. Move the knob on the x-axis to pierce the needle into the egg. If it is difficult to pierce, push the tip of the needle into the egg by tapping the knob with a little finger.
13. Put the solution until the amount of solution at the tip of the needle is about 1/10 the diameter of the egg.
14. Move the knob on the x-axis and bring out the needle.
15. Move the petri dish to move the tip of the needle to another egg. Repeat 12-14 to inject the solution.
16. When changing the direction of the egg, the egg is moved using a manipulating needle.
