Lessons from morpholino-based screening in zebrafish

Abstract

Morpholino oligonucleotides (MOs) are an effective, gene-specific antisense knockdown technology used in many model systems. Here we describe the application of MOs in zebrafish (Danio rerio) for in vivo functional characterization of gene activity. We summarize our screening experience beginning with gene target selection. We then discuss screening parameter considerations and data and database management. Finally, we emphasize the importance of off-target effect management and thorough downstream phenotypic validation. We discuss current morpholino limitations, including reduced stability when stored in aqueous solution. Advances in MO technology now provide a measure of spatiotemporal control over MO activity, presenting the opportunity for incorporating more finely tuned analyses into MO-based screening. Therefore, with careful management, MOs remain a valuable tool for discovery screening as well as individual gene knockdown analysis.

Figure 1:

Workflow using morpholinos as a genetic screening tool. (A) The first step is to identify the target genes to be screened. We identified our target sequences using bioinformatics tools and comparing human, zebrafish and pufferfish genomes. Once the likely start site was identified, the MO was designed using standard MO-mRNA target sequence binding parameters (reviewed in [4]). (B) Second, we established a standard dose curve. We designed 20 MOs against genes with known phenotypes to establish a broad calibration curve for MO effectiveness. (C) We standardized the subsequent screening process as much as possible. (I) For phenotype identification, we used a team-based approach that catered to the individual scientists’ expertise. (II) We used specific biological assays, for example microangiography, to improve the specificity of phenotypic description. (III) We standardized written phenotypic descriptions as much as possible, using PATO-compliant terminology to enhance data communication. (IV) Finally, observed phenotypes were entered into the MODB database for organization, searchability and data sharing. (D) For each phenotype observed with an initial MO, validation was essential to ensure the phenotype was gene-specific and not an off-target effect. (E and F) Once a phenotype was established as gene-specific, that MO could be used to investigate new biology.

Figure 2:

Examples of off-target effects of MOs. (A) A normal wild-type embryo at 24 hpf. The brain is transparent (arrow) and the tail is not bent (arrowhead). The MO used for this demonstration is targeted against SP2054 with and without p53. SP2054 MO shows a nonspecific p53 activation that does not co-localize with the endogenous expression pattern of the targeted gene [17]. (B–E) Examples of the standard toxicity seen in MO injected embryos that are not co-injected with the p53 MO. These four images are placed in order of increasing severity. (B) This embryo demonstrates some neural death, seen as a black area in the brain where it should be transparent (arrow), while the tail is somewhat bent (arrowhead). (C) This embryo shows more neural toxicity (arrows), and the tail is significantly bent (arrowhead). (D) This embryo does not show significant neural toxicity (arrow). However, gnarled tails are commonly noted due to MO toxicity (arrowhead). (E) This embryo shows a classic more severe, ‘monster’ phenotype. This embryo has p53-induced neural toxicity (arrow), but it also shows a very shortened and gnarled tail as well as small head and eyes. (F) Inhibition of p53 can attenuate common off-target effects. Shown is the same SP2054 MO as was used above co-injected with the p53 MO at 3 ng of each MO (therefore 6 ng of total MO). The brain is largely clear with little or no visible neural toxicity (arrow) while the tail remains fairly normal (arrowhead). The monster phenotype can be found in p53 MO co-injected embryos but usually to a lesser extent.