A primer for morpholino use in zebrafish

Abstract

Morpholino oligonucleotides are the most common anti-sense "knockdown" technique used in zebrafish (Danio rerio). This review discusses common practices for the design, preparation, and deployment of morpholinos in this vertebrate model system. Off-targeting effects of morpholinos are discussed as well as method to minimize this potentially confounding variable via co-injection of a tP53-targeting morpholino. Finally, new uses of morpholinos are summarized and contextualized with respect to the complementary, DNA-based knockout technologies recently developed for zebrafish.

FIG. 1.

Morpholino antisense oligonucleotides and mechanisms of gene knockdown. The main antisense chemistry used in zebrafish are morpholino phosphorodiamidate oligonucleotides (morpholinos, MOs). MOs are composed of a phosphorodiamidate backbone with a morpholine ring and the same bases as DNA (A).^, Standard use is through steric hindrance of the normal endogenous splicing (B) or translation (E) mechanisms. MOs targeting the splice donor site inhibit binding of the U1 complex, thus inhibiting lariat formation and incorporation of the intron (C). Inclusion of the intron often leads to premature stops and nonsense-mediated decay of the transcript. MOs targeting the splice donor are proposed to function by preventing binding of the U2AF protein (AF) required to recruit the U2 complex, thereby disrupting lariat formation (D). Translational blocking MOs bind the AUG or 5′ UTR hindering the scanning of the 40S ribosome and block translational inhibition and elongation by the full ribosomal complex (F). Figure adapted from Ekker and Larson.

FIG. 2.

Zebrafish embryo holding trays for injection—The Zebrafish Book design. The process of pouring trays involves the lowering of a plastic mold into molten 1% agarose (A). Special caution needs to be taken to reduce the number of air bubbles that are underneath the tray, as this will disrupt the final rows. Embryo water is added to the tray to lubricate the mold for removal as well as hydrate the tray (B). If a tray is hydrated properly embryos can be kept on the tray for up to 20 min without harm. If the agarose has spilled over the top of the mold, the spatula is used to slice around the edges before removing the tray (C). This leaves an indention in the plate for which to load embryos. Embryo water is left in the tray for a minimum of 5 min to completely hydrate the tray. The final trays (D) are kept up to 2 months at 4°C, thus eliminating evaporation. Up to 40 embryos can be loaded into a row for injections.

FIG. 3.

Microinjector setup and calibration. Quantitation of the amount of solution injected is critical to determining the relevancy of the work. The solution is back-loaded using an Eppendorf microloader pipette tip (A). The needle is then loaded into the shaft (c) of the three-axis micromanipulator (C) to get a proper seal the silicon gasket (b), and metal cap (a) must be added to the needle before insertion (B). To open the tip a number 5 forceps is used to break the tip of the needle (D). Imaging under the highest power of the dissecting microscope helps observe the needle during this process (E). Pressure from the microinjector should be applied at this stage to determine the amount of solution injected. A1-λ capillary is moved toward the tip of the needle gently inserting the tip of the needle into the capillary and applying 10–30 ms pumps from the microinjector (G). Two hands can help keep the capillary steady during this process (F). Embryos are loaded onto the agarose plates. Under high power, move the needle next the embryo with the micromanipulator, quickly insert the needle into the center of the yolk, and inject the proper dosage of solution (H). As a training tool, we utilize a chordin MO with FITC label, so that positive injections can be scored. The phenotype of this training MO is shown in (I). The MO should be distributed ubiquitously throughout the embryo, suggesting a proper delivery and display the chordin phenotype of an expanded blood island—the area just posterior to the yolk sac extension (for a demonstration of this process, see Supplemental Video 1).

FIG. 4.

tP53 knockdown ameliorates nonspecific MO neural death phenotypes. Brightfield images of 1 dpf embryos are shown in panels (A–F). Fluorescent images detecting cellular apoptosis after TUNEL detection are shown in panels (G–L). (B, H) Wnt5 MO1-injected embryos. Note the extensive apoptosis that is ameliorated after co-injection with a tp53 MO (C, I). The resulting embryos show the strong wnt5 mutant phenotype (compare to E, K). (D, J) Not all wnt5-targeted MOs exhibit the off-targeting neural death phenotype. (F, L) The tp53 MO does not block all cell death in the zebrafish embryo. Note the chordin loss of function-induced, tissue-specific apoptosis in chdMO/tp53MO co-injected embryos. Reprinted from Robu et al.