Genetic Tools

Whole Genome Sequence

Using ENU-derived mutagenesis researchers were able to map observable phenotypes to gene functions. This process is called forward genetics. Today the annotated zebrafish genome is available for everybody online. This allows for selection of any genome sequence of interest.

Injection

Injection is the most frequently used technique when working with zebrafish. When injecting, a substance is introduced into the embryo. This is usually done at the early stages of development. This can include substances such as a nucleic acid, protein, or a drug of interest. This technique is the basis of morpholinos and transgenesis.

Morpholinos

Morpholinos are short antisense oligonucleotides and are a way to interfere with a gene’s function. A morpholino is injected into an early embryo to knockdown the expression of a gene of interest. This occurs by the morpholino binding a target sequence and blocking splicing and/or translation of a specific mRNA, resulting in reduced or lost expression of the gene product. These can be used to analyze functions of genes with unknown phenotypes, or reverse genetics.

Transgenesis

Expression plasmids can be injected into the early embryo; similar to the way morpholinos are injected. Depending on what the plasmid specificies, the gene can be specific to a cell type, such as neurons, and be tagged with a fluorescent marker, such as GFP. These tagged genes can be followed throughout development.

Genome Editing

Genome editing allows for sequence-specific manipulation via specifically designed manipulation. This has evolved rapidly in recent times. The first-generation of this technique was using zinc finger nucleases (ZFNs). However recently the CRISPR-Cas system has taken over as the dominant way to do genome editing. This method relies on classic RNA injection protocols. RNA complementary to the genomic target sequence of interest is co-delivered with synthetic capped mRNA encoding Cas9 endonuclease.