Zebrafish Morpholino Gene Knockdown

Zebrafish Morpholino Gene Knockdown

In molecular biology, a Morpholino is a kind of molecule applied to modify gene expression. Morpholinos are commonly used as a research tool for reverse genetics by knocking down gene function. This is achieved by preventing cells from making a targeted protein or by modifying the splicing of pre-mRNA. Knocking down gene expression is a potent method for learning about the function of a particular protein; similarly, causing a specific exon to be spliced out of a protein can help to determine the function of the protein moiety encoded by that exon. Because of their unique chemistry, morpholinos are easy to produce and store, which makes them significantly cost-effective compared to other gene silencing methods.

Illustration of how thestart site (A) and splice site (B) morpholinos work.

Figure 1. Illustration of how thestart site (A) and splice site (B) morpholinos work. (Timme-LaragyA R, et al. 2012)

Compared to mammalian models, zebrafish are relatively easy to manipulate genetically, and there are numerous tools available to do so (such as thegeneration of transgenic and mutant fish, injection of RNA for overexpression, gene targeting through zinc finger nuclease technology). The introduction of antisense morpholino oligonucleotides (MOs) has changed the way that developmental biologists study the mechanics and underlying  molecular mechanisms of embryonic development. MOs have been widely used in studies of zebrafish development, in part due to the lack of methods for  generating targeted null mutants as well as the accessibility of the MO technique to most laboratories. Furthermore, for some applications gene knockdown  offers distinct advantages as compared to gene knockout methods.

Morpholino Gene Knockdown in Zebrafish

In the past few years, the use of MOs to study the mechanistic roles of specific genes in zebrafish embryo toxicity has grown dramatically. Besides, the MO-based screening can create knockdown phenotypes in wild-type embryos, significantly reducing infrastructural needs as compared to a classical mutagenesis screening. Creative Biogene, a zebrafish research company with experienced scientists and advanced technology, can offer  professional zebrafish morpholino gene knockdown services. In cases where a gene is required for development, the MO concentration can be titrated  down to a level that is not embryolethal. Another way to bypass embryolethal gene knockdowns is by the use of "caged" MOs that can be photo-activated  to act in specific locations at specific times, thus generating conditional knockdowns in developing zebrafish.

Workflow of Zebrafish Point Mutation Services

  • Target identification and MO design
  • Microinjection in zebrafish embryos
  • Screening&Validation
  • Assessment of effects

Zebrafish MO knockdown can be applied to confirm the identity of a proposed target protein, in which case the phenotype of the morphant  will mimic the toxic response caused by chemical exposure. Besides, MOs are also excellent tools with which to confirm mechanistic hypotheses generated by exploratory methods. For more information or consultation, please feel free to contact us. Our staff will assist you to get rid of the situation.

References

  • Timme-LaragyA R, et al. Gene knockdown by morpholino-modified oligonucleotides in the zebrafish (Danio rerio) model: applications for developmental toxicology. Developmental Toxicology. Humana Press, Totowa, NJ, 2012: 51-71.
  • Bedell V M, et al. Lessons from morpholino-based screening in zebrafish. Briefings in functional genomics, 2011, 10(4): 181-188.
  • Blum M, et al. Morpholinos: antisense and sensibility. Developmental cell, 2015, 35(2): 145-149.

For research use only. Not intended for any clinical use.

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