The overall goal of this procedure is to inhibit or knock down the expression of a specific protein in the adult zebrafish retina. This is accomplished by first removing the outer cornea of the fish eye. Next, a small hole is created in the inner cornea of the eye.
Then the gene specific morpho is injected into the vitreous. Finally, the morpho is electroporated into the retinal cells. Ultimately, results can be obtained that show reduced target protein expression through either immunofluorescence microscopy of retinal tissue sections, or immuno blotting.
The implications of this technique extend towards therapy of retinal degenerative diseases such as retinitis pigmentosa. Because of the potential to elucidate the underlying mechanisms of Mueller glial cell proliferation in the damaged retina, we may find ways to induce a similar response in the damage to human retina. Though this method can provide insight into retinal regeneration in the adult zebrafish retina, it can be slightly modified to provide information in other processes in the retina, such as the role of specific proteins in photo transduction or visual processing.
Demonstrating the procedure that was developed by Dr.Ryan Tummel while he was a postdoc. In my laboratory will be Dr.Travis Bailey, a current postdoc in my lab. Before working with fish, prepare positively charged lissamine tagged morphos that can be electroporated into cells by diluting 300 NMO of Morpho into 100 microliters of nuclease free water teal to three millimolar solution.
To remove the outer corneal layer, anesthetize six to 12 month old zebrafish in one milligram per milliliter of trica or two feth ethanol in zebrafish tank water wrap zebrafish in a moistened piece of paper towel covering the gills, but leaving the eye exposed. Place the wrapped fish under a stereoscope and increase the magnification until the diameter of the eye fills approximately one third of the visual field. Next, using Dumont number five tweezers, grab the outer cornea near the optic fissure at the site of a slight protrusion or lip to prevent the eye from becoming dislodged.
When removing the cornea, steady it with another set of tweezers. Then pull at a low angle across the eye to remove the cornea. Immediately following the removal of the outer cornea, use a sapphire blade scalpel to make a small incision in the cornea where the pupil meets the iris load.
Point five microliters of the morpho solution into a Hamilton syringe equipped with a 33 gauge removable blunt end needle. Pipe it up and down to remove airable in the line. Carefully insert the needle into the vitreous through the incision.
Do not insert the needle too far to avoid puncturing the retina or displacing the lens. Slowly inject the morpho solution into the vitriol space. Colorless vitreous will leak out of the incision as it is displaced by the morpho solution.
Inject until a small amount of morpho leaks out of the incision. The lissamine tagged morino should be clearly visible inside the eye. Following the injection, return the fish to the tank to recover using the other eye as an unin injected control, set the electroporation parameters to two consecutive 50 millisecond pulses at 75 volts.
With a one second pause between pulses following the injection of approximately 10 fish. Re anesthetize an injected fish and wrap it in a moistened piece of paper. Towel just covering the gills, but leaving the eye exposed.
Transfer the fish to a Petri dish filled with anesthesia. With gloved hands, gently hold the fish to the bottom of the dish filled with anesthesia, taking care to completely submerge the eye. To target the dorsal retina, gently press the positive electrode downward on the ventral half of the eye, causing the eye to rotate and expose the dorsal portion of the eyeball while still pressing down on the ventral side, place the negative electrode 0.5 to one millimeter away from the exposed dorsal half of the eye.
Avoid placing the negative electrode directly on the eye as this will damage the dorsal retina. After determining the appropriate distance between the electrodes, adjust the screw on the handle of the electrodes to maintain a constant spread for subsequent electroporation. Electroporated the eye and return the fish to a tank with fresh tank water to revive immediately subject the fish to a procedure that causes changes in retinal gene expression, such as constant light induced retinal degeneration.
When the retinas are sectioned from nasal to temporal on the dorsal ventral axis, the shaded blue boxes represent the retinal areas that are most often damaged from the electroporation event itself. The shaded pink box represents the area that is targeted for morpho introduction into retinal cells by the electroporation. As shown here in the image on the right three days following the electroporation, the lissamine label is still visualized in all of the layers and cell types in a sectioned retina, including the rod outer segments, the outer nuclear layer, the interclear layer, and the ganglion cell layer.
The blank image on the left is from the unin injected eye, lacking any lissamine tagged morino, the full protein knockdown can be achieved through three to five days following electroporation depending on the efficacy of the morpho. In this example, a negative control morpho or A-P-C-N-A morpho was electroporated into retinas, and the retinas were removed after a one, two or three day exposure to light, the tissue was then processed and immunostain with anti PCNA antibodies. Many PC NA positive cells green were observed in all three control retinas, but PCNA expression was significantly reduced in the morphin retinas.
Once you've mastered this technique, you should be able to perform the procedure on about 20 fish in one hour. While attempting to do this procedure, it is important to remember not to touch the electrodes directly to the eye of the fish following this procedure. Other methods such as immunohistochemistry, situ to hybridization, real time PCR and immuno blotting can be used to answer additional questions such as what genetic pathways are required in retinal regeneration.
