In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

The overall goal of this procedure is to inhibit or knock down the expression of a specific protein in the regenerating adult zebra fish tail fin. This is accomplished by first amputating the tail fin and allowing a small amount of tissue to regenerate. Next morpho solution is micro injected into each blasa on one half of the fin.

The morph pheno is then electroporated into the cells of the blasa, and the other half of the fin is electroporated for control purposes. Finally, the effect of reduced target protein expression on regenerative outgrowth is analyzed. Ultimately, the results show inhibition of tissue regeneration through comparative analysis of the regenerative outgrowth of the injected and non injected halves of the fin.

The implications of this technique extend toward therapy of wound healing and regenerative medicine because of the potential to uncover genes and signaling pathways that are essential to tissue generation. To prepare fluorescein tagged morino dilute 300 nanomoles into 100 microliters of nuclease free water. To make an approximately three millimolar solution, aliquot the solution into multiple paraffin sealed micro centrifuge tubes, and store at room temperature away from light To determine the exact morpho concentration, see the written protocol for details.

Anesthetize adult zebra fish in either trica or two phenoxy ethanol at one milligram per milliliter in tank water. When the fish is fully anesthetized, place it on the clean lid of a Petri dish and using a sterile scalpel or razor blade amputate the fin proximal to the first lipid tracheal branching point, It is important to cut the fin perfectly perpendicular to the anterior posterior plane of the animal because angled cuts will result in uneven fin outgrowth of the dorsal and ventral halves of the fin. Return the fish to the tank and keep it at 33 degrees Celsius to increase the regeneration rate depending on the experimental design weight.

Zero to two days post amputation for fin regeneration. To begin before introducing the morpho, the day before morpho injection, make an injection plate with 2%agarose that has a notch cut out at one end of the well, which will help stabilize the fish for the injection procedure. Just before injecting, dilute the fluorescein tagged morpho and RNAs and DNAs free water to the proper concentration and incubated in a 65 degree Celsius water bath.

For five minutes, load a previously prepared injection needle and cut off the tip at an angle. Next, anesthetize the fish and place it on the injection plate. Remove all excess liquid and place the plate on the stage of a stereo microscope.

Orient the needle just distal to the bony ray. Insert the needle gently into the regenerative tissue just distal to each bony ray and push distally until located in the blastema. When the needle is correctly localized, follow the microinjection system directions and inject a five nanoliter drop of the morpho per injection.

With each injection, a yellow puff of morpho solution can be seen, which helps localize the injection to the blastema working dorsally from the midline. Inject approximately 75 nanoliters of morpho per bony ray on the dorsal side, leaving the ventral side as an electroporation only control immediately following injection of the morpho. Remove the injection plate from the microinjection apparatus.

Fill the injection plate well with the anesthesia solution until the fish is submerged. To ensure that the electroporated electrodes do not touch the fin tissue, rotate the fish on its dorsal side and look straight down the midline for electroporation. Set the electroporation parameters to 10 consecutive 50 millisecond pulses at 15 volts with a one second pause between pulses with three millimeter diameter platinum plate tweezer electrodes set close to but not touching.

The fins electroporated both the dorsal and ventral sides of the fin to prevent charge buildup from bubbles that may form. Wipe the electrodes with a damp kimm wipe after each electroporation. Finally, place the fish on a glass slide or Petri dish and quickly image the fin, making sure to note the dorsal ventral orientation.

This image will be used for regrowth analysis on the following day. Return the fish to the tank if the targeted protein is required for proper fin regeneration. A dramatic difference between the dorsal and ventral halves of the fin should be evident one day post electroporation or three days post amputation.

Take another picture of the fin and match up this image with a corresponding two days post amputation image taken after electroporation. Using NIH image trace the two days post amputation and the three days post amputation areas of both the dorsal and ventral halves. To determine the percent area of dorsal versus ventral fin growth, use the following formula.

The quantity dorsal three days post amputation minus dorsal two days amputation divided by the quantity ventral three days post amputation minus ventral two days post amputation times 100 equals percent area. When assay for fin outgrowth at three days post amputation or 24 hours post electroporation, the fluorescein tagged morino should be present in the dorsal half of the fin. It's normal to see some trailing down to the level of the amputation.

When a control morpho is injected into the dorsal half of the fin 24 hours post electroporation equal outgrowth to that on the ventral side is seen as shown here. Injecting the dorsal half of the fin with an experimental morpho inhibits regrowth on that side. Following this procedure.

Other methods like immunohistochemistry and C two hybridization real-time PCR and immuno blotting can be performed in order to answer additional questions like what genetic pathways are required for regeneration.