The overall goal of this procedure is to create a transgenic zebrafish line that allows the observance of cellular electrical changes during embryogenesis, larval movement, and in tumor genesis. This method could help answer key questions in the developmental biology, physiology, and cancer cell biology fields such as what are the fundamental roles of cellular electrical signaling during embryogenesis and in tumor cells? The main advantage of this technique is that it allows us to track cellular electrical signaling in vivo and in real time.
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 degree 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 five 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 micro injection 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 disposal 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 degree Celsius incubator to let them develop. Check back throughout the day to remove dead fish embryos and replace the water with 0.1%methylene blue in fish water. Around six to eight hours after injection, use 10 injected fish embryos and the hotshot method to prepare genomic DNA.
The following morning, use a dissection microscope with a fluorescent light source to sort out the embryos showing GFP in the non-yolk tissues. These embryos should contain the injected construct. Perform a Tol2 excise assay to check the transposon activity as described previously.
If excised plasma can be detected, keep the injected fish embryos and raise them. Otherwise, repeat the Tol2 mRNA synthesis and micro injection process until achieving positive results from the Tol2 excise assay. To image zebrafish embryos, cross-multiple F2 generation founder fish with wild type fish in individual pairs.
Collect fish embryos at different desired developmental stages according to the zebrafish staging guide. For the early stage fish embryos in fish system water, under a dissection scope, use a pair of forceps to carefully peel and remove the chorions. Use a disposal transfer pipette to transfer a few embryos into a concave glass slide with 3%methylcellulose.
Then using a needle underneath a dissection scope, adjust the embryos to the desired positions to view the cellular GFP activity. For embryos at the less than 12 somite stage, use an epifluorescence compound microscope with a compatible camera and software for imaging. For embryos past the 12 somite stage, use a fluorescence dissection microscope.
To image tumor cell voltage, first identify fish with Malignant Peripheral Nerve Sheath Tumors or MPNST. Place the fish into a 10 centimeter diameter Petri dish and carry out whole mount imaging. Finally following imaging, dissect out the tumor before viewing the tumor cell electrical activity.
In a successful injection, more than 50%of injected embryos will display some degree of GFP in the somatic cells and most of them will show a positive result from the Tol2 transposon excise assay. Here membrane potential changes were examined throughout the cell cycle during zebrafish early embryonic development. As seen in this timelapse video, the cells hyperpolarized before formation of the cleavage furrow.
Moreover, different tissues showed a variety of membrane potentials in one to three-day-old fish embryos. For example, the somite and notocord were generally hyperpolarized compared to the adjacent tissues and organs. In this two-day-old fish embryo, neuromuscular electrical activities are shown during movement with color density changes corresponding to the electric signaling transduction.
Bioelectrical properties of cancer cells were examined in embryos from crosses of ASAP1 reporter fish with an RPL35 mutant which is prone to spontaneous MPNST. Compared to surrounding tissues, tumor cells were more polarized. Once mastered, this micro injection technique can be done in about three hours if performed properly.
While attempting this procedure, it is important to remember that the zebrafish embryos should be in the one cell stage for ideal results. With its development, this technique paves the way for researchers in the field of developmental biology and cell biology to explore in vivo electrical signaling changes in zebrafish and other model organisms. After watching this video, you should have a good understanding of how to create a transgenic zebrafish line by micro injection.
