This method can help answer key questions in the zebrafish heart regeneration field such as the molecular mechanisms regulating heart regeneration. The main advantage of this technique is that this method is very efficient to knock down target gene expression and it's very convenient. To begin this procedure, add 4.2 milliliters of the tricaine stock solution to 100 milliliters aquarium system water to prepare the tricaine working solution.
Then prepare the instruments and materials needed for the surgery which includes a piece of sponge, a Petri dish filled with tricaine working solution, a plastic pipette, a stereo microscope, two sharp forceps, iridectomy scissors, and elbow tweezers. Prepare a small groove in the sponge to hold the zebrafish during the ventricular resection surgery. Next, prepare a recovery tank by filling a three liter zebrafish tank half full with distilled water containing sea salts at a concentration of 60 milligrams per liter.
Using elbow tweezers, transfer an anesthetized fish ventral side up to the sponge groove. Visually locate the margin of the beating heart then use a stereo microscope to locate the heart. Using iridectomy scissors, begin making a small one millimeter incision that penetrates the skin and muscles.
Use forceps tips to carefully tear the silvery epithelial layer of the pericardial sac to expose the heart. Make sure not to insert the forceps too deeply to avoid injuring the heart. Using sharp forceps in the nondominant hand, expose the ventricle by gently holding its apex.
With the other hand, use iridectomy scissors to remove about 20%of the ventricle at the apex. Cover the incision with a wet napkin as the wounds will bleed profusely for 15 to 45 seconds before clotting. After this, put the fish into the recovery tank.
Make sure that the fish regains gill movements within one minute and then resume swimming. If the fish does not show gill movements after 50 seconds in the water, use a plastic pipette to squirt water into the gills until the fish resumes active ventilation. First, dissolve the PAM nanoparticle dendrimer powder in PBS at a concentration of four milligrams per milliliter.
Make aliquots of the solution for one-time use. Dissolve cyanine dyes labeled siRNAs, ALDH1A2 siRNA or scrambled negative control siRNA in distilled water at a concentration of 10 micromolar. To begin preparing the siRNA-encapsulated nanoparticle solution, mix siRNA with an equal volume of dendrimer solution.
Incubate at room temperature for 20 minutes. Next, prepare a piece of grooved sponge, a 10 centimeter Petri dish filled with tricaine solution, a plastic pipette, a stereo microscope, elbow tweezers, and an insulin syringe. Prepare a fresh recovery tank by filling a three liter zebrafish tank half full with distilled water containing sea salts at a concentration of 60 milligrams per liter.
Using the elbow tweezers, transfer an anesthetized fish ventral side up to the groove in the moist sponge. Locate the beating heart under the stereo microscope. Load 10 microliters of the siRNA nanoparticle solution into the insulin syringe avoiding bubbles as much as possible.
Using the elbow tweezers in the nondominant hand, hold the thoracic region to immobilize the fish. In the other hand, hold the insulin syringe at a 30 degree angle and inject the solution into the thoracic cavity. After this, transfer the fish to the injection recovery tank.
Prepare a microcentrifuge tube containing one milliliter of 4%paraformaldehyde solution for each group. After anesthetizing the fish, place is supinely into the groove of a moist sponge. Make a large incision in the thoracic region and use tweezers to open the incision widely.
Next, use the tweezers to grip the outflow tract and carefully pull out the whole heart. Rinse the heart with PBS using a napkin to rapidly remove any extra liquid. Transfer up to 10 hearts into one of the 1.5 milliliter microcentrifuge tubes containing paraformaldehyde.
Gently invert the tube several times. Then store overnight at four degrees Celsius. The next day, use the in vivo imaging system to measure the fluorescence intensity of Cy5-siRNAs injected hearts.
Repeat this measurement using three to four zebrafish hearts from each group. First, pour liquid nitrogen into an insulated container filling up a third of the container's volume. Transfer an anesthetized fish supinely into the groove of a moist sponge.
After dissecting the hearts as previously described, remove the outflow tract and atrium. Use the tweezers to transfer the ventricles into a 1.5 milliliter microcentrifuge tube. Immediately place the tube into the insulated container with liquid nitrogen.
Let the tubes float on the liquid nitrogen for a few minutes. Then use tweezers to remove the tubes. Repeat this process until all of the ventricles are collected making sure to use between six and 10 hearts for each group.
After this, use an RNA isolation reagent to extract the total RNA from the hearts. Assess the expression levels of the respective genes by quantitative RT-PCR as outlined in the text protocol. In this study, the efficiency of dendrimer-mediated siRNA delivery is determined by respecting the apex of the zebrafish heart ventricle.
The fluorescence signal is detectable in the hearts injected with dendrimer-encapsulated Cy5-siRNA at three, 24, and 48 hours post-injection. However, it is hardly detectable in the hearts from the mock and naked groups at 48 hours post-injection. This suggests that the PAMAM nanoparticle dendrimer effectively facilitates the delivery of siRNAs into the zebrafish heart and is stable for at least two days.
Zebrafish are then microinjected with dendrimer-encapsulated siRNA to investigate its effect on gene silencing. The mRNA expression level of the target gene is seen to decrease in hearts treated with the corresponding dendrimer-encapsulated siRNA. Hearts treated with dendrimer-encapsulated scrambled siRNA however did not show a decrease in their mRNA expression level.
This demonstrates that PAMAM nanoparticles dendrimer-mediated siRNAs achieve gene-specific silencing in adult zebrafish hearts. Once mastered, this technique can be done in about one hour if it is performed properly. While attempting this procedure, it's important to remember to successfully make ventricular resection.
Following this procedure, other methods like western blots, immunohistochemical staining, or other functional analysis can be performed in order to answer additional questions like the function of target genes. After its development, this technique paved the way for researchers in the field of zebrafish to explore regeneration in heart. Don't forget that working with paraformaldehyde and RNA isolation reagent can be extremely hazardous and precautions such as masks should always be taken while performing this procedure.
