What is Morpholino zebrafish?

Morpholino oligonucleotides (MOs) are an effective, gene-specific antisense knockdown technology used in many model systems. Here we describe the application of MOs in zebrafish (Danio rerio) for in vivo functional characterization of gene activity.

Why are morpholinos used in zebrafish?

A neutrally charged phosphorodiamidate backbone results in molecules with high binding affinity for RNA, thereby facilitating steric hindrance of proper transcript processing or translation. Two types of MO applications in zebrafish include splice blocking13 and translational blocking.

What is Morpholino knockdown?

A Morpholino, also known as a Morpholino oligomer and as a phosphorodiamidate Morpholino oligomer (PMO), is a type of oligomer molecule (colloquially, an oligo) used in molecular biology to modify gene expression. Gene knockdown is achieved by reducing the expression of a particular gene in a cell.

How does Morpholino work?

Unlike many antisense types (e.g. siRNA, phosphorothioates), Morpholinos generally do not cause degradation of their RNA targets; instead, they block the biological activity of the target RNA until that RNA is degraded naturally, which releases the Morpholino.

How long do Morpholinos last?

They are normally injected into the yolk and affect every cell in the embryo, so may compromise normal development if the disrupted protein is required ubiquitously. The effects also only last up to five days, making them unsuitable for studying gene function at later stages of development.

How long is an oligonucleotide?

Oligonucleotides are small molecules 8–50 nucleotides in length that bind via Watson-Crick base pairing to enhance or repress the expression of target RNA.

What are antisense morpholino oligonucleotides?

Morpholino oligonucleotides are nonionic DNA analogs available from Gene Tools LLC [10,11]. They possess altered backbone linkages compared with DNA or RNA (Figure 1). In spite of their altered backbone, morpholinos bind to complementary nucleic acid sequences by Watson-Crick base-pairing.

How does siRNA affect gene expression?

The siRNA-induced post transcriptional gene silencing starts with the assembly of the RNA-induced silencing complex (RISC). The complex silences certain gene expression by cleaving the mRNA molecules coding the target genes. This cleavage results in mRNA fragments that are further degraded by cellular exonucleases.

What are morpholino oligonucleotides useful for?

Caged morpholino oligonucleotides (cMOs) are useful research tools in developmental biology because they allow spatiotemporal control of gene expression in whole organisms.

Are oligonucleotide primers?

Oligonucleotides made up of 2′-deoxyribonucleotides are the molecules used in polymerase chain reaction (PCR). These are referred to as primers and are used to massively amplify a small amount of DNA.

What is NT in genetics?

Background: Increased nuchal translucency (NT) is an important biomarker associated with increased risk of fetal structural anomalies. It is known to be contributed by a wide range of genetic etiologies from single-nucleotide variants to those affecting millions of base pairs.

How do antisense oligonucleotides work?

Antisense oligonucleotides intervene at a critical intermediate stage between DNA and proteins – where the DNA is converted into a molecule called messenger RNA (or mRNA for short). mRNA is very similar to DNA, but much less stable, and chemically very slightly different. It acts as the template for making proteins.

What is morpholino zebrafish?

How does a morpholino knockdown gene expression?

Why are zebrafish used in research?

Zebrafish embryos are nearly transparent which allows researchers to easily examine the development of internal structures. Zebrafish have a similar genetic structure to humans. They share 70 per cent of genes with us. 84 per cent of genes known to be associated with human disease have a zebrafish counterpart.

What does a morpholino do?

Morpholinos are nucleotide analogs that recognize and bind short sequences (about 25 nucleotides) at the transcription start site or at splice sites of pre-mRNAs, and thus block the translation or proper splicing of the mRNA (Summerton & Weller, 1997).

How do you knock down a gene?

RNA interference (RNAi) is a means of silencing genes by way of mRNA degradation. Gene knockdown by this method is achieved by introducing small double-stranded interfering RNAs (siRNA) into the cytoplasm. Small interfering RNAs can originate from inside the cell or can be exogenously introduced into the cell.

Can zebrafish be aggressive?

Despite the fact that zebrafish is a gregarious species that forms shoals, when allowed to interact in pairs, both males and females express aggressive behavior and establish dominance hierarchies.

Are zebrafish poisonous?

The results showed that prothioconazole exposure to zebrafish embryos produced a series of toxic symptoms, including hatching inhibition, shortening of body length, pericardial cyst and yolk cyst. In addition, prothioconazole exposure caused significant lipid peroxidation and oxidative damage.

Why does a target RNA morpholino hybrid not promote mRNA degradation?

Because the RNA is degraded, any sequence within the coding region of the target gene has the potential to be a useful antisense site. Morpholinos, by contrast, form RNA-morpholino hybrids that are not substrates for RNase H, and thus the mRNA is not degraded.

What is difference between knockdown and knockout?

The main difference between gene knockout and knockdown is that gene knockout involves the complete erasing of target genes, or inactivating them through nonsense mutations whereas gene knockdown leads to abortive protein translation and degradation of that mRNA.

How are Morpholino oligomers used in zebrafish?

Morpholino oligomers have been used widely and for many years in the zebrafish community to transiently knock down the function of target genes. It has often been difficult, however, to reliably discriminate between specific and non-specific effects, and thus generally accepted guidelines to control for morpholino side effects do not exist.

Are there off target effects of MOS in zebrafish?

However, concerns soon arose about the off-target effects of MOs, motivating leading scientists in the zebrafish and Xenopus fields to publish a set of guidelines including controls and rescue experiments that, if followed carefully, would help distinguish specific phenotypes from off-target effects [ 3 ].

How is Morpholino used to disrupt gene function?

However, it quickly became a priority to be able to disrupt the function of specific genes in a targeted way.

How are antisense oligonucleotides used in zebrafish?

Antisense MO oligonucleotides are composed of synthetically modified nucleotides and are designed to bind mRNA transcripts. MOs may be designed to sterically inhibit the translation or splicing of mRNA transcripts and have been widely exploited in zebrafish and frogs as a reverse genetic tool for knocking down gene function (Heasman, 2002 ).