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, m