Tol2 Transposon-Mediated Zebrafish Transgenesis: A Procedure to Generate Transgenic Zebrafish Following the Coinjection of Tol2 System Into Fertilized Zebrafish Embryos
Trascrizione
After preparing Tol2 transposase mRNA and injection solution, according to the text protocol, the afternoon before injection, set up four to six breeding tanks with at least two males and two females. To reduce the amount of fish waste, and to induce a breeding response, avoid feeding the fish in the afternoon.
The following morning, remove the prepared injection solution from the negative 80 degrees Celsius freezer, and place it on ice. Pull the dividers in the fish breeding tanks and allow the fish to mate. In general, fish lay eggs within 20 to 30 minutes.
While waiting, using a micropipette puller with the following parameters, pull needles from capillary glass. Use a laboratory wipe to break the end of the needle and create a beveled edge. A smaller diameter is preferred for decreasing embryo mortality.
Once the fish have laid eggs, collect them in a 10-centimeter diameter Petri dish. Immediately under the dissection microscope, remove all abnormal embryos and fish waste. Then, pipette the fertilized embryos into a prepared 3% agarose injection mold. Remove excess water to help keep the embryos in place.
Once all of the rows are filled with viable embryos, arrange them so that the single cells are all oriented at a 45-degree angle horizontally with respect to the needle. This will make injection much easier later. Next, while wearing gloves, use a 20-microliter loading pipette tip to remove 5 microliters of the prepared construct from the tube on ice.
Carefully insert the pipette tip into the back end of the broken capillary tube to where it begins to taper, to get the reagent as close to the tip as possible, and expel it into the capillary. If there are still air bubbles, shake the needle, making sure not to break the tip.
Insert the needle straight into the microinjection needle holder, and carefully tighten until the needle stays in place, then, adjust the angle to about 45 degrees. Once the needle is prepared and attached, turn on the microscope and gas pressure tank. Adjust the injection volume by using approximately 0.5 PSI for holding, and 30 PSI for ejection.
Check that the solution ejects from the needle when pressing the pedal. Using a stage micrometer with a drop of mineral oil, adjust the volume and flow of the solution to approximately 10 micrometers in diameter. Ensure that the back pressure allows a small amount of solution to drip out of the needle.
If there is not enough back pressure, capillary action will cause liquid to enter the needle, and destroy the mRNA. Once the needle is calibrated, insert the needle into the single cell of the fertilized embryos by using the edge of the gel-notch to provide a backing that keeps the embryo in place, and allows the needle to apply pressure without moving the embryo.
Once the tip of the needle is in the single cell, press the pedal to release the desired amount of solution. It is important to inject the solution into the cell, not the yolk for generating transgenic zebrafish. Repeat this process for all of the embryos.
When completed, use a disposable 3.4-milliliter transfer pipette and fish system water to transfer the injected embryos into a labeled dish by rinsing them out of the agarose notch. Store the embryos in a 28.5 degrees Celsius incubator to let them develop.
