Any MO-induced phenotype must be validated to confirm that it is due to gene-specific effects. Several standard protocols for validating a putative morphant phenotype have been established (for detailed validation methods, see [3
]). First, a second MO of independent (and typically non-overlapping) sequence can be designed against the target transcript. A splice-site targeting MO [3
] is now often used for this purpose because it allows the phenotype to be correlated with knockdown efficacy through quantitative measurements of altered or reduced transcript levels via quantitative real-time PCR (qRT–PCR). However, the second MO can also be a translation-blocking MO with a different sequence than the first. Regardless of the type of MO used for validation, the purpose of this experiment is to determine whether the resulting phenotypes overlap, as would be expected for independent MOs targeted to the same gene. For known genes with available antibodies, it is possible to validate efficacy with western blotting or related immunohistochemical methods to correlate reduced protein levels of the targeted gene with an observed phenotype (D).
RNA rescue is another valuable validation method: the MO of interest and mRNA of the targeted gene (free of the MO targeting sequence) are co-injected and embryos are examined for amelioration of the gene-specific phenotype. For many genes with localized expression, MO knockdown followed by ubiquitous mRNA delivery seldom results in truly wild-type animals. Instead, the expectation is for rescue of a specific pathway or biological process within the more comprehensive in vivo setting.
If a mutant is available in a gene of interest, the morphant and mutant phenotypes should be compared. Validation is thus addressed where the two phenotypes agree. However, several issues may complicate a direct correlation between mutant and morphant. First, translation-blocking MOs can uncover maternally provided gene activity [6
] resulting in phenotypes not noted in zygotic-only homozygous mutant animals. For some loci, generating combined maternal/zygotic mutations can circumvent this potential concern. Second, many mutations may only be hypomorphic in nature, with the resulting homozygous animals retaining some gene function. One way to address this latter concern is to conduct low-dose MO injections into heterozygous mutant embryos and compare those against injections into wild-type sibling embryos. The mutant genetic background should be permissive for the MO phenotype of interest.
We also assessed the uniqueness of the observed phenotype with respect to the overall collection. For example, an observed phenotype with a signature effect on specific aspects of biology when compared to the collected results from the hundreds of other MOs was used as corroborating evidence that the phenotype in question is likely gene-specific [13
]. For example, using this criterion in the secretome screen, 26/150 putative gene targets yielded likely gene-specific knockdowns [13
], whereas 16 of 64 targets tested in the more focused hematopoietic screen yielded likely genes necessary for blood development [22
]. In contrast, when multiple MOs induce the same phenotype, the result is potentially an off-target effect. This correlated dataset operates as an ongoing internal control.