DNA construct preparation
1. Thaw the required plasmids for this experiment. ATV+ and ATV- can be used for constitutive activation while optoATV+ and optoATV- are useful for optogenetic control of the cargo transport. The cargo needs to be tagged by GFP (or any other variant that can be bound by GBP) at either the N- or C- terminus. Include the Piggybac transposon for stable integration into the genome to suit long-term activation.
2. Dilute the DNA constructs in DPBS to reach a final concentration of 1-2 mg/ml.
We recommend to test different molar ratios of the motor:cargo plasmids at the beginning. Start around 1:1. The optimal ratio may vary with different constructs.
3. Combine with phenol red solution to reach a final concentration of around 0.1 mM to aid visualization.
4. Make a capillary glass microinjection needle using a standard needle puller.
5. Backfill the glass needle with 5 μl of DNA injection solution using a microloader tip.
Axolotl electroporation
1. Prepare 0.25 g/L Tricaine solution by dissolving Tricaine powder into pre-chilled Holtfreter's solution.
2. Use Tris solution to adjust the pH of the solution to the range of 6-8.
3. Anesthetize the axolotl by placing it in the Tricaine solution for approximately 8 minutes, until the animal is no longer responsive to motion.
4. Place the axolotl on its belly in an agarose-filled petri dish.
5. Observe the axolotl under a standard desktop microscope.
6. Stretch out and position the forelimbs with sterile tweezers to enable injection and the subsequent electroporation.
7. Pressure-inject the DNA injection solution into the axolotl forelimb, approximately 1-2 mm distal to the elbow joint. Use 2-3 different injection sites to make sure the solution is fully diffused to different parts of the limb.
We recommend using multiple small pulses to inject the solution so that the site of injection can be more carefully controlled.
8. Pour pre-chilled DPBS solution in the petri dish to immerse the injected limb.
9. Place an electrode tweezer on both sides of the injected forelimb with the distance between the electrodes set at 2-4 mm apart.
10. Apply three poring pulses of 150 V and 5 ms with 10 ms interval followed by five transfer pulses of 50 V and 50 ms with 950 ms interval. Keep the forelimb in place during the electroporation with a tweezer by holding the proximal part of the forelimb. These parameters can be adjusted for different applications.
11. Amputate the most distal region of the forelimb using a sterile surgical scalpel.
Amputation can be performed on different days before or after electroporation.
12. Place the axolotl back to Holtfreter's solution for recovery for at least 3 days before imaging.
Live axolotl confocal imaging
1. Prepare 0.25 g/L Tricaine solution by dissolving Tricaine powder into pre-chilled Holtfreter's solution.
2. Use Tris solution to adjust the pH of the solution to the range of 6-8.
3. Anesthetize the axolotl by placing it in the Tricaine solution for approximately 8 minutes, until the animal is no longer responsive to motion.
4. Cut a sterile hybrislip to approximately 0.8 x 0.8 cm in size, and attach four small particles of adhesive (we used Blu-Tack) to the four corners of the hybrislip using a sterile tweezer.
5. Place the anesthetized axolotl inside a sterile fluorodish on its belly, and remove major liquid droplets inside the dish.
6. Stretch out the electroporated and amputated forelimb using a sterile tweezer.
7. Mount the prepared hybrislip onto the blastema region, with the least possible force applied with the tweezer to each corner of the hybrislip. Make sure the hybrislip stays in place without deforming the blastema.
8. Fill the fluorodish with the Tricaine solution to submerge the axolotl.
9. Perform standard confocal imaging on the mounted live axolotl at the site of the blastema. Make sure to use the least possible laser power for imaging. Do not image for longer than 30 minutes to minimize the impact of long-term anesthesia on the animal, or submerge the animal in Holtfreter's solution for a brief recovery after 30 minutes before continuing imaging.
10. For optogenetic experiments, perform the anesthesia and mounting strictly in the dark. We used a red flashlight to aid the process. Use short pulses of 488 nm laser to activate optoATVs.