Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

The zebrafish are housed in an AAALAC-approved animal facility, and all experiments were carried out according to the protocols approved by the Purdue Animal Care and Use Committee (PACUC).

1. Tol2 Transposon Plasmid Construct Preparation

NOTE: Tol2, a transposon that was discovered in medaka fish, has widely been used in the zebrafish research community^8,9. It has been successfully adopted to the Gateway site-specific recombination-based cloning system and known as the Tol2 kit¹⁰. The Tol2 kit allows for a more convenient way of creating customized expression constructs, while also increasing the efficiency of transgenesis. Thus, it was an easy decision to take advantage of this system, and create a ubiquitous ASAP1 expression zebrafish line using a validated ubiquitin promoter to drive ASAP1¹¹.

1. Creating a middle entry ASAP1 construct: pDONR221-ASAP1
   1. Acquire the genetically encoded voltage sensor ASAP1 construct, pcDNA3.1/Puro-CAG-ASAP1 (Plasmid#52519), from Addgene. To amplify the ASAP1 coding region, set up a PCR using the customized primers (attB1-ASAP1F and attB2-ASAP1R) flanked with attB sequences at the 5' end of the primers (Figure 1). Phusion DNA polymerase was chosen for its high efficiency, and PCR conditions were optimized based on the previously published protocol¹².
   2. Load the 50 µL PCR products into a 1% TAE gel using a regular pipette with 200 µL tips, and perform electrophoresis at 160 V in a horizontal gel tank for about 30 min.
   3. Check the gel under a UV transilluminator (353 nm), excise out the desired band using a blade/scalpel under a UV transilluminator as previously published^13,14, and put the DNA containing gel sample into a clean 1.5 mL microcentrifuge tube.
   4. Perform gel purification for the ASAP1 PCR products. Recover the DNA in the excised gel using a commercial DNA gel purification kit following manufacturer's instruction, and elute the DNA into 20 µL of water. Take 1 µL as a sample, and measure the DNA concentration using a spectrophotometer. Flow the software instructions using water as a blank control¹⁵.
   5. Take 100 ng of purified PCR product and mix it with 150 ng of pDONR221 plasmid in 10 µL of TE buffer (10 mM Tris, 1 mM EDTA pH 8.0)¹⁶. Add 2 µL of BP Clonase II into the reaction and incubate the reaction at room temperature overnight.
   6. On the second day, add 1 µL of proteinase K into the reaction and incubate the reaction at 37 °C for 30 min.
   7. Perform transformation. Transfer the reaction and mix it with 50 µL of Top10 competent E. coli cells and incubate the cells on ice for 30 min. Then, transfer the reaction tube into a 42 °C water bath for 1 min. Immediately remove the tube and incubate on ice for 2 min. Next, put the tube into a 37 °C shaker and incubate it for 1 h.
   8. Take the tube out and plate the cells onto a kanamycin LB plate. Next, incubate the plate overnight (16-18 h) at 37 °C.
   9. Pick single and well-separated colonies and inoculate them into 14 mL cell culture tubes with 3 mL of LB medium. Culture them overnight at 37 °C in a shaker with a rotation speed of 250 rpm (rotation per minute).
   10. Perform miniprep using a commercial miniprep kit following its instruction manual¹⁷.
   11. Sequence 3-4 plasmids with Sanger sequencing to identify positive pDONR221-ASAP1 clones using M13F and M13R sequencing primers.
2. Creating the Tol2 construct for microinjection: pDestTol2-ubi-ASAP1
   1. Choose the sequencing verified pDONR221-ASAP1 clone and measure its DNA concentration using a spectrophotometer following the software instruction using water as a blank control¹⁵.
   2. Take 100 µg of pDONR221-ASAP1 and mix it with 100 µg of Tol2 kit 5-end plasmid (pENTR5'_ubi, Addgene #27320), 100 µg of p3E-polyA (Tol2 kit #302) and 150 µg of pDestTol2pA2 (Tol2 kit #394). Adjust total volume to 8 µL using TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) in a 1.5 mL microcentrifuge tube and mix well by a 2-5 s brief vortex. Then, add 2 µL of LR Clonase II plus, and incubate the reaction at room temperature overnight.
   3. On the second day, add 1 µL of proteinase K into the reaction using a 10 µL pipette, and incubate the reaction at 37 °C for 30 min.
   4. Perform transformation and identify positive clones as described above (steps 1.1.7-11).
   5. Measure the DNA concentration of sequencing verified pDestTol2-ubi-ASAP1 clone (Figure 1B) using a spectrophotometer¹⁵. Usually the concentration is around 200ng/µL.

2. Prepare Tol2 Transposase mRNA and Injection Solution

1. Streak E. coli glycerol stock of pCS2FA-transposase plasmid (Tol2 kit #396) onto a LB plate (with 100 µg/mL ampicillin) using a sterilized inoculation loop. Incubate the plate at 37 °C in an incubator overnight. The next day, pick a single colony and inoculate it into 3 mL of LB (100 µg/mL ampicillin) using a sterilized 10 µL pipette tip. Culture it overnight at 37 °C in a shaker with a rotation speed of 250 rpm.
2. Perform miniprep on the E. coli culture using a commercial miniprep kit following its instruction manual. Elute plasmid DNA into 30-50 µL of TE buffer by 1-minute centrifuge at 14,000 rpm, and measure its DNA concentration with a spectrophotometer.
3. Linearize 1-2 µg of plasmid with Not I endonuclease and purify the DNA with a DNA cleaning kit following its instruction manual after Not I digestion. Elute the DNA into 5 µL of water by centrifuging at 14,000 rpm for 1 minute. The expected concentration is about 200-300 ng/µL.
4. Perform in vitro transcription with Not I linearized pCS2FA-transposase as a DNA template using a commercial SP6 transcription kit.
5. Once the reaction is finished, purify Tol2 transposase mRNA using a commercial RNA cleaning kit following the manufacturer's instructions. Finally, elute mRNA into 20 µL RNAase-free water and measure the RNA concentration in a spectrophotometer. The expected concentration is about 1-3 µg/µL. Samples can be stored in a -80 °C freezer if needed.
6. Prepare the microinjection solution by mixing 20 ng/µL pDestTol2-ubi-ASAP1 and Tol2 transposase mRNA (100 ng/µL) in a microcentrifuge tube by pipetting. To prevent nucleic acid degradation caused by repeated thawing and refreezing, aliquot 6 µL per tube and store it in a -80 °C freezer for future use.

3. 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 (pDestTol2-ubi-ASAP1 construct and 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. Check back throughout the day removing dead fish embryos and replace water with 0.1% methylene blue in fish water.
16. Around 6-8 hours after injection, take 10 individual injected fish embryos and prepare genomic DNA from them using the Hotshot method¹⁸.
17. The following morning, use a dissection microscope with a fluorescence light source to sort out the embryos showing GFP in the non-yolk tissues. These fish embryos should contain the injected construct.
18. Perform Tol2 excise assay to check the transposon activity as described previously¹⁹. If excised plasmid can be detected, keep the injected fish embryos and raise them. If no excised plasmid can be detected, repeat the Tol2 mRNA synthesis and microinjection process until achieving the positive results of the Tol2 excise assay.

4. Establish Transgenic ASAP1 fish, Tg(ubi:ASAP1)

1. Raise the injected fish (F₀ generation) to adulthood as described previously in the zebrafish book²⁰. This usually takes about 4 months.
2. Take a single adult F₀ fish and cross it with a single opposite gender wildtype fish. Collect fish embryos after breeding later in the day. Keep the collected fish embryos in the 28.5 °C incubator in fish water with 0.1% methylene blue.
3. On the third day, check the fish embryos underneath a fluorescent dissection microscope with a GFP filter. Sort out green fish embryos, if there are any, and raise them to adulthood as F₁ generation transgenic fish, Tg(ubi:ASAP1).
   NOTE: Mendelian ratio is not expected since most of the parental F₀ fish are germ-line genetic chimeras.
4. Cross single F₁ adult fish with wildtype fish and collect fish embryos. Sort out green fish embryos and raise them to adulthood as F₂ generation fish.
   NOTE: Green and non-green fish embryos should be close to 1:1 if there is a single transgene.
5. To view electric potential changes in tumor like malignant peripheral nerve sheath tumors (MPNST), cross the F₂ generation Tg(ubi:ASAP1) fish with rpl35^hi258/wt fish. It is known that visible tumors start to be found in the 6-8 month old adults^21,22.

5. Imaging

1. To image zebrafish embryos, take multiple F₂ generation founder fish and cross them with wildtype fish in individual pairs. Collect fish embryos at different desired developmental stages according to the zebrafish staging guide²³.
2. For the early stages of fish embryos, peel and remove chorions of the embryos carefully using a pair of forceps under a dissection scope in a 10-cm diameter Petri dish with fish system water.
3. Transfer a few fish embryos onto a concaved glass slide with 3% methylcellulose using a 3.4 mL disposable transfer pipette. Adjust the embryos to the desired positions to view the cellular GFP activity using a needle underneath a fluorescent dissection scope.
4. For the moving stages of fish embryos (older than 12 somite stage), use 0.05% tricaine mesylate to anesthetize the fish embryos before transferring them to slides. Briefly, fish embryos were emerged into 0.05% tricaine mesylate in fish water until they stopped swimming and loss body balance. Also, add a drop of 0.05% tricaine mesylate with the methylcellulose on the slide.
5. For less than 12 somite stage fish embryos, use an epi-fluorescence compound microscope with a compatible camera and software for imaging. For older than 12 somite stage fish embryos, use a fluorescence dissection microscope.
6. To image tumor cell voltage, first identify the fish with MPNST tumors. Then, anesthetize the fish with 0.05% tricaine mesylate. For whole mount imaging, put the fish into a 10-cm diameter Petri dish. To view the tumor cell electrical activity, fish tumors may be dissected out after whole mount imaging.