Morpholino Injection: A Method to Knockdown Target Gene Expression to Study Function in Zebrafish

- One approach to determine the function of a gene is to knock down its expression and observe the effect. Morpholino oligomers are often used in knock-down studies in zebrafish. A morpholino is a synthetic DNA-like molecule around 25 bases long, which inhibits target gene expression.

For morpholino treatment, place an anesthetized fish with a partially amputated tail fin on an injection plate and position the plate under a stereo microscope. Only inject the dorsal side while the uninjected ventral side acts as a control. Morpholinos can function in two ways depending on the sequence they target. First, they combine to the translation start site on an mRNA and prevent protein synthesis by blocking translation.

Second, it can bind to the intron-exon junction on a pre-mRNA and prevent proper splicing, resulting in a defective transcript that will be degraded by the cell. Later, examine the caudal fins for outgrowth. Differences between regrowth of the morpholino-treated and untreated regions indicate that the targeted protein plays a role in fin regeneration. In this protocol, we will use morpholinos as part of a caudal fin regeneration assay.

- The day before morpholino 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 fluoroscein-tagged morpholino in RNAase and DNAase free water to the proper concentration and incubate it in a 65 degrees 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 morpholino per injection. With each injection, a yellow puff of morpholino solution can be seen, which helps localize the injection to the blastema. Working dorsally from the midline, inject approximately 75 nanoliters of morpholino per bony ray on the dorsal side, leaving the ventral side as an electroporation-only control.