Tol2 Transposon-Mediated Zebrafish Transgenesis: A Procedure to Generate Transgenic Zebrafish Following the Coinjection of Tol2 System Into Fertilized Zebrafish Embryos

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Microinjection

1. Set up 4-6 breeding tanks with at least 2 males and 2 females the afternoon before injection. These fish must not be fed in the afternoon. This step will reduce the amount of fish waste and save time to clean them out the next morning, while also helping to induce a breeding response.
2. The following morning, remove the prepared injection solution comprising of: (i) a Tol2 transposon construct containing a zebrafish ubiquitin promoter and the fluorescent voltage indicator gene Accelerated Sensor of Action Potentials 1 (ASAP1), and (ii) Tol2 mRNA, from the -80 °C freezer, and place it on ice.
3. Pull the dividers in the fish breeding tanks and allow the fish to mate. In general, fish lay eggs within 20-30 minutes after pulling out the divider. If not, wait 1-2 hours longer. Some fish may not lay eggs at all. In this case, repeat this experiment for fish embryo collection.
4. While waiting, make sure there are needles prepared with the tip broken at an angle creating a beveled edge with forceps, or by breaking on a delicate task wiper.
   1. Pull needles from capillary glass on a micropipette puller using the following parameters: heat 545; pull 60; velocity 80; time 250; pressure 500. Break the needle with forceps underneath a dissection scope (with eye-piece ruler for diameter estimation) by holding the forceps at an angle approximately 45°. Desired needle diameters can be variable depending on the microinjector settings, but smaller diameter is preferred for decreasing embryo mortality.
5. Once the fish have laid eggs, collect them in a 10-cm diameter Petri dish and bring them to the dissection scope. Remove all abnormal embryos and fish waste.
6. Pipet the fertilized embryos into the prepared 3% agarose injection mold. Remove excess water to help keep the embryos in place.
7. Once all of the rows are filled with viable embryos, arrange them so that the single cells all face the same direction toward the needle, which is about a 45° angle horizontally. This will make injection much easier later.
8. While wearing gloves, use a 20 μL loading pipet tip and remove 5 μL of the prepared construct from the tube on ice.
9. Carefully insert the tip into the back end of the broken capillary tube all the way to where it begins to tapper, as to get the reagent as close to the tip. If there are still air bubbles, shake the needle, making sure to not break the tip.
10. Insert the needle straight into the microinjection needle holder and carefully tighten until the needle stays in place. Adjust the angle to about 45°.
11. Once the needle is prepared and attached, turn on the microscope and gas pressure tank. Commercial CO₂ tanks are generally good for this purpose. The injection volume is adjusted by the holding and ejection pressure: approximate 0.5 psi for holding and 30 psi for ejection. Be sure to check that the solution comes out when pressing the pedal.
12. Using a stage micrometer with a drop of mineral oil, adjust the volume and flow of the solution to ~150 μm in diameter (about 2 nL). Ensure that the back pressure will let a small amount 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.
13. Once the needle is calibrated, begin injecting the construct into the single cell of the fertilized embryos.
    NOTE: This takes a large amount of practice, patience, and finesse, due to the cell membrane being hard to pierce. It is important to inject the solution into the cell, not the yolk, for generating transgenic zebrafish. This is different from morpholino injection. It does not matter which side the needle enters the cell as long as the construct goes in the cell. The transgenesis will have a very low rate of success if injected into the yolk instead of the cell. Single-cell stage injection is also important, or somatic chimera fish will be created. This will reduce the chance of the transgene going into to the germ cells.
14. Use 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. Repeat this process for all of the embryos.
15. When completed, transfer the injected embryos into a labeled dish by rinsing them out of the agarose notch with fish system water and a disposable 3.4 mL transfer pipette. Store the embryos in a 28.5 °C incubator to let them develop.

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자료

Name	Company	Catalog Number	Comments
Agarose RA	Amresco	N605-500G	For making the injection gels
Bright field dissection scope	Nikon	SMZ 745	Dechorionation, microinjection, mounting
Disposable transfer pipette 3.4 ml	Thermo Scientific	13-711-9AM	Fish embryos and water transfer
Forceps #5	WPI	500342	Dechorionation and needle breaking
Loading tip	Eppendorf	930001007	For loading injection solution into capilary needles
Microinjection mold	Adaptive Science Tools	TU-1	To prepare agaorse mold tray for holding fish embryos during injection
Microinjector	WPI	Pneumatic Picopump PV820	Microinjection injector
Micro-manipulator	WPI	Microinjector MM3301R	Microinjection operation
Micropipette puller	Sutter instrument	P-1000	For preparing capillary needle
Mineral oil	Amresco	J217-500ml	For calibrating injection volume
Plastic Petri dishes	VWR	25384-088	For holding fish or fish embryos during imaging process
Stage Micrometer	Am Scope	MR100	Microinjection volume calibration
Thin wall glass capillaries	WPI	TW100F-4	Raw glass for making cappilary needle