Morpholino-based knock-downs
ZeClinics’ knock-down (KD) approaches are a fast and very easy way to see if your gene of interest might have a phenotype (reverse genetic analysis), and warrant making a germline mutant.
Our genetic experts can generate functional KDs of gene expression in F0 animals employing morpholino oligonucleotides (MOs). This procedure does not compromise the genomic DNA but alters the transcribed mRNA.
Transient gene KD will help establish a direct link between the biochemical function of a gene product and its role in vivo. Phenotyping is possible with good controls but the decreasing concentration of MOs leads to sub-optimal KD usually by 4dpf.
The main difference of this technique compared to other alternatives for fast target validation, such as Crispants, is the transient nature of the genetic modulation.
Applications
- Rapid and economic technique for reverse genetic analysis: seeking to find what phenotypes are controlled by particular genes.
- Fast target validation: screening of several candidate genes.
Advantages
Scalable solution: high throughput screening of multiple candidate genes
Confirmation of the phenotypic output before establishing the KO line
Non-invasive, in vivo readouts to detect KO-induced phenotypes at the level of cells, tissues or organs
Method description
MOs bind the target mRNAs and act either by altering its splicing or by blocking its translation. As a result of the injection of MOs into one-cell-stage embryos, the synthesis of the proteins encoded by the targeted mRNA is blocked or compromised. The resulting phenotypes are transient, as larvae tend to recover as MOs get degraded. For this reason, MOs-based approaches are better suited to study phenotypes arising during the early stages of embryonic development (<5 dpf).
Figure 1. Mode of action of MOs blocking the synthesis of the proteins encoded by the target mRNAs. Splice blocking morpholinos alter the process of splicing of pre-mRNA, while translation blocking MOs impede protein translation.
Readouts
Results of the phenotypic analysis according to customers' interests and needs:
- Teratogenic phenotypes
- Organ-specific morphological or functional defects (heart-specific, liver-specific, etc.)
- Behavioral phenotypes
We'd like to hear from you
If you want more information about our MO-based
knock-down services or have any other questions,
please contact our experts.
References
- Van Gils M, Vanakker OM. Morpholino-Mediated Gene Knockdown in Zebrafish: It Is All About Dosage and Validation. J Invest Dermatol. 2019 Jul;139(7):1599-1600.