ZeGenesis – Genetic Services

BY
OUR
PLATFORMS

morpholinos_knock_down_icon Morpholino-based knock-downs

Transient gene knock-down (KD) via morpholino oligonucleotides (MOs) is one of the most rapid and economical techniques for performing reverse genetic analysis. Reverse genetics is a method of molecular genetics that seeks to find what phenotypes are controlled by particular genes.

ZeClinics genetic experts can generate functional KDs of gene expression in F0 animals employing MOs, which block the translation or splicing of mRNAs and inhibit protein synthesis. This procedure does not compromise the genomic DNA but alters the transcribed mRNA.

KD approaches 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. It is a fast and very easy way to see if your gene of interest might have a phenotype and warrant making a germline mutant.

Applications

  • Temporary inhibition of gene expression at early developmental stages
  • Cost-effective screening of candidate genes that are suspected to be involved in a given disease

Advantages

Fast target validation prior to the generation of stable loss-of-function alleles

High throughput screening of multiple candidate genes

Non-invasive, in vivo readouts to detect KD-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 impedes 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

References

  1. 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.