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120-766: This article discusses only the Morpholino antisense oligomers, which are nucleic acid analogs . The word "Morpholino" can occur in other chemical names, referring to chemicals containing a six-membered morpholine ring. To help avoid confusion with other morpholine-containing molecules, when describing oligos "Morpholino" is often capitalized as a trade name , but this usage is not consistent across scientific literature. Morpholino oligos are sometimes referred to as PMO (for phosphorodiamidate morpholino oligomer), especially in medical literature. Vivo-Morpholinos and PPMO are modified forms of Morpholinos with chemical groups covalently attached to facilitate entry into cells. Gene knockdown

240-432: A homozygous embryo. The zebrafish embryos are transparent and they develop outside of the uterus, which allows scientists to study the details of development starting from fertilization and continuing throughout development. The zebrafish is also demonstrably similar to mammalian models and humans in toxicity testing, and exhibits a diurnal sleep cycle with similarities to mammalian sleep behavior. However, zebrafish are not

360-412: A phosphate backbone, a pentose sugar, either ribose or deoxyribose , and one of four nucleobases . An analogue may have any of these altered. Typically the analogue nucleobases confer, among other things, different base pairing and base stacking properties. Examples include universal bases, which can pair with all four canonical bases, and phosphate-sugar backbone analogues such as PNA , which affect

480-605: A solute carrier that appeared to be required for melanin production, and confirmed its function with a Morpholino knockdown. The orthologous gene was then characterized in humans and a one base pair difference was found to strongly segregate fair-skinned Europeans and dark-skinned Africans. Zebrafish with the nacre mutation have since been bred with fish with a roy orbison (roy) mutation to make Casper strain fish that have no melanophores or iridophores, and are transparent into adulthood. These fish are characterized by uniformly pigmented eyes and translucent skin. Transgenesis

600-507: A (d5SICS–dNaM) complex mimicking the natural (dG–dC) base pair. One of the most common base analogs is 5-bromouracil (5BU), the abnormal base found in the mutagenic nucleotide analog BrdU. When a nucleotide containing 5-bromouracil is incorporated into the DNA, it is most likely to pair with adenine; however, it can spontaneously shift into another isomer which pairs with a different nucleobase , guanine . If this happens during DNA replication,

720-740: A DNA duplex is the hope to obtain nanoscopic self-assembling metal wires, though this has not been realized yet. An unnatural base pair (UBP) is a designed subunit (or nucleobase ) of DNA that is created in a laboratory and does not occur in nature. In 2012, a group of American scientists led by Floyd Romesberg, a chemical biologist at the Scripps Research Institute in San Diego, California, published that his team had designed two unnatural base pairs named d5SICS and dNaM . More technically, these artificial nucleotides bearing hydrophobic nucleobases feature two fused aromatic rings that form

840-433: A connected metal-base pair. This motif does not accommodate stacked Hg in a duplex due to an intrastrand hairpin formation process that is favored over duplex formation. Two thymines across from each other do not form a Watson-Crick base pair in a duplex; this is an example where a Watson-Crick basepair mismatch is stabilized by the formation of the metal-base pair. Another example of a metal complexing to natural nucleobases

960-423: A consequence of the loss of the targeted protein and not a consequence of the knockdown oligo type. It appears that these effects are sequence-specific; as in most cases, if a Morpholino is associated with non-target effects, the 4-base mismatch Morpholino will not trigger these effects. A cause for concern in the use of Morpholinos is the potential for "off-target" effects. Whether an observed morphant phenotype

1080-410: A d5SICS–dNaM complex or base pair in DNA. In 2014, the same team reported that they had synthesized a plasmid containing natural T-A and C-G base pairs along with the best-performing UBP Romesberg's laboratory had designed and inserted it into cells of the common bacterium E. coli , which successfully replicated the unnatural base pairs through multiple generations. This is the first known example of

1200-485: A dedicated online database of genetic, genomic, and developmental information, the Zebrafish Information Network (ZFIN). The Zebrafish International Resource Center (ZIRC) is a genetic resource repository with 29,250 alleles available for distribution to the research community. D. rerio is also one of the few fish species to have been sent into space . Research with D. rerio has yielded advances in

1320-488: A flexible arm, presumably extruding from the major groove of the helix. Due to low processivity of the nucleotides linked to bulky adducts such as florophores by [Taq polymerase]s, the sequence is typically copied using a nucleotide with an arm and later coupled with a reactive fluorophore (indirect labelling): Fluorophores find a variety of uses in medicine and biochemistry. The most commonly used and commercially available fluorescent base analogue, 2-aminopurine (2-AP), has

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1440-734: A fluorescence quantum yield of approximately 0.2 both in single- and in double-strands irrespective of surrounding bases. Also the oxo-homologue of tC called tC (both commercially available), 1,3-diaza-2-oxophenoxazine, has a quantum yield of 0.2 in double-stranded systems. However, it is somewhat sensitive to surrounding bases in single-strands (quantum yields of 0.14–0.41). The high and stable quantum yields of these base analogues make them very bright, and, in combination with their good base analogue properties (leaves DNA structure and stability next to unperturbed), they are especially useful in fluorescence anisotropy and FRET measurements, areas where other fluorescent base analogues are less accurate. Also, in

1560-461: A general-purpose tool for blocking interactions of proteins or nucleic acids with mRNA. Morpholinos have become a standard knockdown tool in animal embryonic systems, which have a broader range of gene expression than adult cells and can be strongly affected by an off-target interaction. Following initial injections into frog or fish embryos at the single-cell or few-cell stages, Morpholino effects can be measured up to five days later, after most of

1680-412: A guanine will be inserted as the opposite base analog, and in the next DNA replication, that guanine will pair with a cytosine. This results in a change in one base pair of DNA, specifically a transition mutation . Additionally, nitrous acid (HNO2) is a potent mutagen that acts on replicating and non-replicating DNA. It can cause deamination of the amino groups of adenine, guanine and cytosine. Adenine

1800-514: A high-fluorescence quantum yield free in solution (0.68) that is considerably reduced (appr. 100 times but highly dependent on base sequence) when incorporated into nucleic acids. The emission sensitivity of 2-AP to immediate surroundings is shared by other promising and useful fluorescent base analogues like 3-MI, 6-MI, 6-MAP, pyrrolo-dC (also commercially available), modified and improved derivatives of pyrrolo-dC, furan-modified bases and many other ones (see recent reviews). This sensitivity to

1920-538: A journal article and in book form. Morpholinos are in development as pharmaceutical therapeutics targeted against pathogenic organisms such as bacteria or viruses and genetic diseases . A Morpholino-based drug eteplirsen from Sarepta Therapeutics received accelerated approval from the US Food and Drug Administration in September 2016 for the treatment of some mutations causing Duchenne muscular dystrophy , although

2040-579: A laboratory animal was pioneered by the American molecular biologist George Streisinger and his colleagues at the University of Oregon in the 1970s and 1980s; Streisinger's zebrafish clones were among the earliest successful vertebrate clones created. Its importance has been consolidated by successful large-scale forward genetic screens (commonly referred to as the Tübingen/Boston screens). The fish has

2160-467: A large variety of plastic items, disrupt the endocannabinoid system and thereby affect reproduction in a sex-specific manner. Zebrafish feeding practices vary significantly across different developmental stages, reflecting their changing nutritional needs. For newly hatched larvae, which begin feeding at approximately 5 days post-fertilization (dpf), small live prey such as Paramecium or rotifers are commonly used until they reach 9–15 dpf. This early diet

2280-462: A living organism passing along an expanded genetic code to subsequent generations. This was in part achieved by the addition of a supportive algal gene that expresses a nucleotide triphosphate transporter which efficiently imports the triphosphates of both d5SICSTP and dNaMTP into E. coli bacteria. Then, the natural bacterial replication pathways use them to accurately replicate the plasmid containing d5SICS–dNaM. The successful incorporation of

2400-422: A methyl-7-guanosine), and several bases of rRNAs (methylated). Often, tRNAs are heavily modified postranscriptionally in order to improve their conformation or base pairing, in particular in or near the anticodon: inosine can base pair with C, U, and even with A, whereas thiouridine (with A) is more specific than uracil (with a purine). Other common tRNA base modifications are pseudouridine (which gives its name to

2520-414: A mutant strain (though compensation will obscure a phenotype in some mutants), by testing the Morpholino in a null mutant background to detect additional phenotypic changes or by dominant-negative methods. As mentioned above, rescue of observed phenotypes by coinjecting a rescue mRNA is, when feasible, a reliable test of specificity of a Morpholino. For a Morpholino to be effective, it must be delivered past

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2640-460: A new strain of zebrafish, named Casper, whose adult bodies had transparent skin. This allows for detailed visualization of cellular activity, circulation, metastasis and many other phenomena. In 2019 researchers published a crossing of a prkdc and a IL2rga strain that produced transparent, immunodeficient offspring, lacking natural killer cells as well as B - and T-cells . This strain can be adapted to 37 °C (99 °F) warm water and

2760-580: A number of commercially available global platforms exist for analysis of both gene expression by microarrays and promoter regulation using ChIP-on-chip . The Wellcome Trust Sanger Institute started the zebrafish genome sequencing project in 2001, and the full genome sequence of the Tuebingen reference strain is publicly available at the National Center for Biotechnology Information (NCBI)'s Zebrafish Genome Page . The zebrafish reference genome sequence

2880-470: A sequence of bases in DNA , is converted into the structure of a protein. A Morpholino can modify splicing, block translation, or block other functional sites on RNA depending on the Morpholino's base sequence. Bound to the 5'-untranslated region of messenger RNA (mRNA), Morpholinos can interfere with progression of the ribosomal initiation complex from the 5' cap to the start codon. This prevents translation of

3000-412: A specialised protein , known as fibroblast growth factor , to ensure their spinal cords heal without glial scarring after injury. In addition, hair cells of the posterior lateral line have also been found to regenerate following damage or developmental disruption. Study of gene expression during regeneration has allowed for the identification of several important signaling pathways involved in

3120-520: A specific metal. For example, a nucleoside modified with a pyridine-2,6-dicarboxylate has shown to bind tightly to Cu , whereas other divalent ions are only loosely bound. The tridentate character contributes to this selectivity. The fourth coordination site on the copper is saturated by an oppositely arranged pyridine nucleobase. The asymmetric metal base pairing system is orthogonal to the Watson-Crick base pairs. Another example of an artificial nucleobase

3240-645: A target sequence within an RNA, inhibiting molecules that might otherwise interact with the RNA. Morpholino oligos are often used to investigate the role of a specific mRNA transcript in an embryo . Developmental biologists inject Morpholino oligos into eggs or embryos of zebrafish , African clawed frog ( Xenopus ), sea urchin and killifish ( F. heteroclitus ) producing morphant embryos, or electroporate Morpholinos into chick embryos at later development stages. With appropriate cytosolic delivery systems, Morpholinos are effective in cell culture . Vivo-Morpholinos, in which

3360-647: A third base pair for replication and transcription. Afterward, Ds and 4-[3-(6-aminohexanamido)-1-propynyl]-2-nitropyrrole (Px) was discovered as a high fidelity pair in PCR amplification. In 2013, they applied the Ds-Px pair to DNA aptamer generation by in vitro selection (SELEX) and demonstrated the genetic alphabet expansion significantly augment DNA aptamer affinities to target proteins. The possibility has been proposed and studied, both theoretically and experimentally, of implementing an orthogonal system inside cells independent of

3480-630: A third base pair is a significant breakthrough toward the goal of greatly expanding the number of amino acids which can be encoded by DNA, from the existing 20 amino acids to a theoretically possible 172, thereby expanding the potential for living organisms to produce novel proteins . Earlier, the artificial strings of DNA did not encode for anything, but scientists speculated they could be designed to manufacture new proteins which could have industrial or pharmaceutical uses. Transcription of DNA containing unnatural base pairs and translation of corresponding mRNA were actually achieved recently. In November 2017,

3600-512: A universally ideal research model; there are a number of disadvantages to their scientific use, such as the absence of a standard diet and the presence of small but important differences between zebrafish and mammals in the roles of some genes related to human disorders. Zebrafish have the ability to regenerate their heart and lateral line hair cells during their larval stages. The cardiac regenerative process likely involves signaling pathways such as Notch and Wnt ; hemodynamic changes in

3720-440: A yolk with a single enormous cell on top (see image, 0 h panel), which divides into two (0.75 h panel) and continues dividing until there are thousands of small cells (3.25 h panel). The cells then migrate down the sides of the yolk (8 h panel) and begin forming a head and tail (16 h panel). The tail then grows and separates from the body (24 h panel). The yolk shrinks over time because the fish uses it for food as it matures during

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3840-422: Is 16,596 base pairs. This is within 100 base pairs of other related species of fish, and it is notably only 18 pairs longer than the goldfish ( Carassius auratus ) and 21 longer than the carp ( Cyprinus carpio ). Its gene order and content are identical to the common vertebrate form of mitochondrial DNA. It contains 13 protein -coding genes and a noncoding control region containing the origin of replication for

3960-520: Is a derived member of the genus Brachydanio , of the family Cyprinidae . It has a sister-group relationship with Danio aesculapii . Zebrafish are also closely related to the genus Devario , as demonstrated by a phylogenetic tree of close species. The zebrafish is native to freshwater habitats in South Asia where it is found in India, Pakistan, Bangladesh, Nepal and Bhutan. The northern limit

4080-444: Is a capacity of explicit memory systems, typically associated with conscious experience . The Mauthner cells integrate a wide array of sensory stimuli to produce the escape reflex . Those stimuli are found to include the lateral line signals by McHenry et al. 2009 and visual signals consistent with looming objects by Temizer et al. 2015, Dunn et al. 2016, and Yao et al. 2016. The approximate generation time for Danio rerio

4200-794: Is a nucleobase analogue, 7-deaza-GTP and is used to sequence CG rich regions, instead 7-deaza-ATP is called tubercidin , an antibiotic. It has been suggested that the RNA world may have been preceded by an "RNA-like world" where other nucleic acids with a different backbone, such as GNA , PNA , and TNA existed, however, evidence for this hypothesis been called "tenuous". Naturally occurring bases can be divided into two classes according to their structure: Artificial nucleotides ( Unnatural Base Pairs (UBPs) named d5SICS UBP and dNaM UBP ) have been inserted into bacterial DNA but these genes did not template mRNA or induce protein synthesis. The artificial nucleotides featured two fused aromatic rings which formed

4320-489: Is a popular approach to study the function of genes in zebrafish. Construction of transgenic zebrafish is rather easy by a method using the Tol2 transposon system. Tol2 element which encodes a gene for a fully functional transposase capable of catalyzing transposition in the zebrafish germ lineage. Tol2 is the only natural DNA transposable element in vertebrates from which an autonomous member has been identified. Examples include

4440-495: Is a spotted colour morph of the zebrafish which arose due to a pigment mutation. Xanthistic forms of both the zebra and leopard pattern, along with long-finned strains, have been obtained via selective breeding programs for the aquarium trade. Various transgenic and mutant strains of zebrafish were stored at the China Zebrafish Resource Center (CZRC), a non-profit organization, which was jointly supported by

4560-405: Is able to integrate into most cells of the embryo. A known problem with gene knockdowns is that, because the genome underwent a duplication after the divergence of ray-finned fishes and lobe-finned fishes , it is not always easy to silence the activity of one of the two gene paralogs reliably due to complementation by the other paralog. Despite the complications of the zebrafish genome ,

4680-426: Is achieved by reducing the expression of a particular gene in a cell. In the case of protein-coding genes, this usually leads to a reduction in the quantity of the corresponding protein in the cell. Knocking down gene expression is a method for learning about the function of a particular protein; in a similar manner, causing a specific exon to be spliced out of the RNA transcript encoding a protein can help to determine

4800-633: Is annotated as part of the Ensembl project , and is maintained by the Genome Reference Consortium . In 2009, researchers at the Institute of Genomics and Integrative Biology in Delhi, India, announced the sequencing of the genome of a wild zebrafish strain, containing an estimated 1.7 billion genetic letters. The genome of the wild zebrafish was sequenced at 39-fold coverage. Comparative analysis with

4920-502: Is capable of laying 200–300 eggs in one morning in approximately 5 to 10 at time. Male zebrafish are furthermore known to respond to more pronounced markings on females, i.e., "good stripes", but in a group, males will mate with whichever females they can find. What attracts females is not currently understood. The presence of plants, even plastic plants, also apparently encourages spawning. Exposure to environmentally relevant concentrations of diisononyl phthalate (DINP), commonly used in

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5040-510: Is crucial for their growth and survival, as these small organisms provide essential nutrients. As the larvae develop, from 15 dpf onwards, they are typically transitioned to a diet that includes brine shrimp nauplii and dry feeds, which are more nutritionally balanced and easier to manage in laboratory settings. For larvae aged 25 dpf, feeding rates can range from 50% to 300% of their body weight (BW) per day, depending on their size and growth requirements. As zebrafish grow into juveniles (30–90 dpf),

5160-406: Is deaminated to hypoxanthine , which base pairs to cytosine instead of thymine. Cytosine is deaminated to uracil, which base pairs with adenine instead of guanine. Deamination of guanine is not mutagenic. Nitrous acid-induced mutations also are induced to mutate back to wild-type. Commonly fluorophores (such as rhodamine or fluorescein ) are linked to the ring linked to the sugar (in para) via

5280-674: Is debated, but there are several unused possibilities. Furthermore, adenine is not the most stable choice for base pairing: in Cyanophage S-2L, diaminopurine (DAP) is used instead of adenine. Diaminopurine basepairs perfectly with thymine as it is identical to adenine but has an amine group at position 2 forming 3 intramolecular hydrogen bonds, eliminating the major difference between the two types of basepairs (weak A-T vs strong C-G). This improved stability affects protein-binding interactions that rely on those differences. Other combination include: However, correct DNA structure can form even when

5400-401: Is distinguished from naturally occurring DNA or RNA by changes to the backbone of the molecule. However, the polyelectrolyte theory of the gene proposes that a genetic molecule require a charged backbone to function. In May 2014, researchers announced that they had successfully introduced two new artificial nucleotides into bacterial DNA, and by including individual artificial nucleotides in

5520-422: Is due to the intended knockdown or an interaction with an off-target RNA can often be addressed in embryos by running another experiment to confirm that the observed morphant phenotype results from the knockdown of the expected target. This can be done by recapitulating the morphant phenotype with a second, non-overlapping Morpholino targeting the same mRNA, by confirmation of the observed phenotypes by comparing with

5640-517: Is entirely based on pre-1930 records and likely refers to close relatives only described later, notably Danio kyathit . Likewise, old records from Sri Lanka are highly questionable and remain unconfirmed. Zebrafish have been introduced to California, Connecticut, Florida and New Mexico in the United States, presumably by deliberate release by aquarists or by escape from fish farms . The New Mexico population had been extirpated by 2003 and it

5760-807: Is in the South Himalayas , ranging from the Sutlej river basin in the Pakistan–India border region to the state of Arunachal Pradesh in northeast India. Its range is concentrated in the Ganges and Brahmaputra River basins, and the species was first described from Kosi River (lower Ganges basin) of India. Its range further south is more local, with scattered records from the Western and Eastern Ghats regions. It has frequently been said to occur in Myanmar (Burma), but this

5880-418: Is joined there by various other eukaryotic initiation factors , forming the initiation complex. The initiation complex scans along the mRNA strand until it reaches a start codon , and then the large subunit of the ribosome attaches to the small subunit and translation of a protein begins. This entire process is referred to as gene expression; it is the process by which the information in a gene , encoded as

6000-493: Is linked to male infertility. The researchers cloned oestrogen-sensitive genes and injected them into the fertile eggs of zebrafish. The modified fish turned green if placed into water that was polluted by oestrogen. In 2015, researchers at Brown University discovered that 10% of zebrafish genes do not need to rely on the U2AF2 protein to initiate RNA splicing . These genes have the DNA base pairs AC and TG as repeated sequences at

6120-446: Is that with hydroxypyridone nucleobases, which are able to bind Cu inside the DNA duplex. Five consecutive copper-hydroxypyridone base pairs were incorporated into a double strand, which were flanked by only one natural nucleobase on both ends. EPR data showed that the distance between copper centers was estimated to be 3.7 ± 0.1 Å, while a natural B-type DNA duplex is only slightly larger (3.4 Å). The appeal for stacking metal ions inside

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6240-429: Is the formation of A-Zn-T and G-Zn-C at high pH; Co and Ni also form these complexes. These are Watson-Crick base pairs where the divalent cation in coordinated to the nucleobases. The exact binding is debated. A large variety of artificial nucleobases have been developed for use as metal base pairs. These modified nucleobases exhibit tunable electronic properties, sizes, and binding affinities that can be optimized for

6360-451: Is three months. A male must be present for ovulation and spawning to occur. Zebrafish are asynchronous spawners and under optimal conditions (such as food availability and favorable water parameters) can spawn successfully frequently, even on a daily basis. Females are able to spawn at intervals of two to three days, laying hundreds of eggs in each clutch . Upon release, embryonic development begins; in absence of sperm, growth stops after

6480-407: Is to reduce gene expression or modify splicing using Morpholino antisense technology. Morpholino oligonucleotides (MO) are stable, synthetic macromolecules that contain the same bases as DNA or RNA; by binding to complementary RNA sequences, they can reduce the expression of specific genes or block other processes from occurring on RNA. MO can be injected into one cell of an embryo after

6600-409: Is unclear if the others survive, as the last published records were decades ago. Elsewhere the species has been introduced to Colombia and Malaysia. Zebrafish typically inhabit moderately flowing to stagnant clear water of quite shallow depth in streams, canals, ditches, oxbow lakes , ponds and rice paddies . There is usually some vegetation, either submerged or overhanging from the banks, and

6720-682: Is usually difficult, though there are a few systems allowing useful uptake of unmodified Morpholino oligos (including uptake into muscle cells with Duchenne muscular dystrophy or the vascular endothelial cells stressed during balloon angioplasty ). Though they permeate through intercellular spaces in tissues effectively, unconjugated PMOs have limited distribution into the cytosol and nuclear spaces within healthy tissues following IV administration. Systemic delivery into many cells in adult organisms can be accomplished by using covalent conjugates of Morpholino oligos with cell-penetrating peptides , and, while toxicity has been associated with moderate doses of

6840-414: Is very rapid, and its embryos are relatively large, robust, and transparent, and able to develop outside their mother. Furthermore, well-characterized mutant strains are readily available. Other advantages include the species' nearly constant size during early development, which enables simple staining techniques to be used, and the fact that its two-celled embryo can be fused into a single cell to create

6960-576: The Carp (including Grass Carp, Ctenopharyngodon idella ) and Milkfish ( Chanos chanos ) which are quite close, both being in the Cyprinidae . However it should also be noted that Danio consistently proves to be a useful model for mammals in many cases and there is dramatically more genetic distance between them than between Danio and any farmed fish. In a glucocorticoid receptor -defective mutant with reduced exploratory behavior , fluoxetine rescued

7080-622: The Ministry of Science and Technology of China and the Chinese Academy of Sciences . The Zebrafish Information Network ( ZFIN ) provides up-to-date information about current known wild-type (WT) strains of D. rerio , some of which are listed below. Hybrids between different Danio species may be fertile: for example, between D. rerio and D. nigrofasciatus . D. rerio is a common and useful scientific model organism for studies of vertebrate development and gene function. Its use as

7200-769: The TΨC loop ), dihydrouridine (which does not stack as it is not aromatic), queuosine, wyosine, and so forth. Nevertheless, these are all modifications to normal bases and are not placed by a polymerase. Canonical bases may have either a carbonyl or an amine group on the carbons surrounding the nitrogen atom furthest away from the glycosidic bond, which allows them to base pair (Watson-Crick base pairing) via hydrogen bonds (amine with ketone, purine with pyrimidine). Adenine and 2-aminoadenine have one/two amine group(s), whereas thymine has two carbonyl groups, and cytosine and guanine are mixed amine and carbonyl (inverted in respect to each other). The precise reason why there are only four nucleotides

7320-472: The cell membrane into the cytosol of a cell. Once in the cytosol, Morpholinos freely diffuse between the cytosol and nucleus, as demonstrated by the nuclear splice-modifying activity of Morpholinos observed after microinjection into the cytosol of cells. Different methods are used for delivery into embryos, into cultured cells or into adult animals. A microinjection apparatus is usually used for delivery into an embryo, with injections most commonly performed at

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7440-425: The central nervous system and somite tissues of zebrafish embryos. Most of these effects are due to activation of p53 -mediated apoptosis and can be suppressed by co-injection of an anti-p53 Morpholino along with the experimental Morpholino. Moreover, the p53-mediated apoptotic effect of a Morpholino knockdown has been phenocopied using another antisense structural type, showing the p53-mediated apoptosis to be

7560-432: The replication crisis in biomedical research. Genetic differences in wild-type lines among populations maintained at different research institutions have been demonstrated using both Single-nucleotide polymorphisms and microsatellite analysis. Due to their fast and short life cycles and relatively large clutch sizes, D. rerio or zebrafish are a useful model for genetic studies. A common reverse genetics technique

7680-427: The 3' hydroxyl group normally present in DNA and therefore cannot bond with the next base. The lack of the 3' hydroxyl group terminates the chain reaction as the DNA polymerases mistake it for a regular deoxyribonucleotide. Another chain-terminating analogue that lacks a 3' hydroxyl and mimics adenosine is called cordycepin . Cordycepin is an anticancer drug that targets RNA replication. Another analogue in sequencing

7800-410: The 32-cell stage, reducing gene expression in only cells descended from that cell. However, cells in the early embryo (less than 32 cells) are permeable to large molecules, allowing diffusion between cells. Guidelines for using Morpholinos in zebrafish describe appropriate control strategies. Morpholinos are commonly microinjected in 500pL directly into 1–2 cell stage zebrafish embryos. The morpholino

7920-462: The Watson-Crick hydrogen bonds are replaced by the interaction between a metal ion with nucleosides acting as ligands. The possible geometries of the metal that would allow for duplex formation with two bidentate nucleosides around a central metal atom are tetrahedral , dodecahedral , and square planar . Metal-complexing with DNA can occur by the formation of non-canonical base pairs from natural nucleobases with participation by metal ions and also by

8040-399: The absence of an immune system makes the use of patient derived xenografts possible. In January 2013, Japanese scientists genetically modified a transparent zebrafish specimen to produce a visible glow during periods of intense brain activity. In January 2007, Chinese researchers at Fudan University genetically modified zebrafish to detect oestrogen pollution in lakes and rivers, which

8160-460: The approval process was mired in controversy. Other Morpholino-based drugs golodirsen , viltolarsen , and casimersen (also for Duchenne muscular dystrophy) were approved by the FDA in 2019–2021. Morpholino oligos were conceived by Summerton ( Gene Tools ) at AntiVirals Inc. (now Sarepta Therapeutics) and originally developed in collaboration with Weller. Morpholinos are synthetic molecules that are

8280-434: The artificial interaction produced between LEF1 and Catenin beta-1 /β-catenin/ CTNNB1 . Dorsky et al. 2002 investigated the developmental role of Wnt by transgenically expressing a Lef1/β-catenin reporter. There are well-established protocols for editing zebrafish genes using CRISPR-Cas9 and this tool has been used to generate genetically modified models. In 2008, researchers at Boston Children's Hospital developed

8400-547: The bacteria to synthesize "unnatural" proteins. Another demonstration of UBPs were achieved by Ichiro Hirao's group at RIKEN institute in Japan. In 2002, they developed an unnatural base pair between 2-amino-8-(2-thienyl)purine (s) and pyridine-2-one (y) that functions in vitro in transcription and translation, for the site-specific incorporation of non-standard amino acids into proteins. In 2006, they created 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa) as

8520-424: The bases are not paired via hydrogen bonding; that is, the bases pair thanks to hydrophobicity, as studies have shown with DNA isosteres (analogues with same number of atoms) such as the thymine analogue 2,4-difluorotoluene (F) or the adenine analogue 4-methylbenzimidazole (Z). An alternative hydrophobic pair could be isoquinoline and pyrrolo[2,3-b]pyridine Other noteworthy basepairs: In metal base-pairing,

8640-452: The borders of introns on a strand of pre-mRNA, or by blocking the nucleophilic adenine base and preventing it from forming the splice lariat structure, or by interfering with the binding of splice regulatory proteins such as splice silencers and splice enhancers . Preventing the binding of snRNP U1 (at the donor site) or U2 / U5 (at the polypyrimidine moiety and acceptor site) can cause modified splicing , commonly excluding exons from

8760-434: The bottom is sandy, muddy or silty, often mixed with pebbles or gravel. In surveys of zebrafish locations throughout much of its Bangladeshi and Indian distribution, the water had a near-neutral to somewhat basic pH and mostly ranged from 16.5 to 34 °C (61.7–93.2 °F) in temperature. One unusually cold site was only 12.3 °C (54.1 °F) and another unusually warm site was 38.6 °C (101.5 °F), but

8880-416: The cellular genetic material in order to make a completely safe system, with the possible increase in encoding potentials. Several groups have focused on different aspects: Zebra danio The zebrafish ( Danio rerio ) is a freshwater fish belonging to the minnow family ( Cyprinidae ) of the order Cypriniformes . Native to South Asia, it is a popular aquarium fish , frequently sold under

9000-444: The coding region of the targeted transcript (called " knocking down " gene expression ). This is useful experimentally when an investigator wishes to know the function of a particular protein; Morpholinos provide a convenient means of knocking down expression of the protein and learning how that knockdown changes the cells or organism. Some Morpholinos knock down expression so effectively that, after degradation of preexisting proteins,

9120-651: The culture media, were able to passage the bacteria 24 times; they did not create mRNA or proteins able to use the artificial nucleotides. The artificial nucleotides featured 2 fused aromatic rings. Several nucleoside analogues are used as antiviral or anticancer agents. The viral polymerase incorporates these compounds with non-canonical bases. These compounds are activated in the cells by being converted into nucleotides, they are administered as nucleosides since charged nucleotides cannot easily cross cell membranes. Nucleic acid analogues are used in molecular biology for several purposes: Ribose 's 2' hydroxy group reacts with

9240-769: The damaged heart are sensed by ventricular endothelial cells and their associated cardiac cilia by way of the mechanosensitive ion channel TRPV4 , subsequently facilitating the Notch signaling pathway via KLF2 and activating various downstream effectors such as BMP-2 and HER2/neu . In 2011, the British Heart Foundation ran an advertising campaign publicising its intention to study the applicability of this ability to humans, stating that it aimed to raise £50 million in research funding. Zebrafish have also been found to regenerate photoreceptor cells and retinal neurons following injury, which has been shown to be mediated by

9360-424: The dedifferentiation and proliferation of Müller glia . Researchers frequently amputate the dorsal and ventral tail fins and analyze their regrowth to test for mutations. It has been found that histone demethylation occurs at the site of the amputation, switching the zebrafish's cells to an "active", regenerative, stem cell-like state. In 2012, Australian scientists published a study revealing that zebrafish use

9480-492: The detrimental effects of inbreeding depression . Inbreeding depression is predominantly caused by the homozygous expression of recessive deleterious alleles. For zebrafish, inbreeding depression might be expected to be more severe in stressful environments, including those caused by anthropogenic pollution . Exposure of zebrafish to environmental stress induced by the chemical clotrimazole, an imidazole fungicide used in agriculture and in veterinary and human medicine, amplified

9600-448: The effects of inbreeding on key reproductive traits. Embryo viability was significantly reduced in inbred exposed fish and there was a tendency for inbred males to sire fewer offspring. Zebrafish are common models for research into fish farming , including pathogens and parasites causing yield loss or spreading to adjacent wild populations. This usefulness is less than it might be due to Danio ' s taxonomic distance from

9720-480: The end of the caudal fin . Its shape is fusiform and laterally compressed, with its mouth directed upwards. The male is torpedo -shaped, with gold stripes between the blue stripes; the female has a larger, whitish belly and silver stripes instead of gold. Adult females exhibit a small genital papilla in front of the anal fin origin. The zebrafish can reach up to 4–5 cm (1.6–2.0 in) in length, although they typically are 1.8–3.7 cm (0.7–1.5 in) in

9840-457: The ends of each intron . On the 3'ss (3' splicing site), the base pairs adenine and cytosine alternate and repeat, and on the 5'ss (5' splicing site), their complements thymine and guanine alternate and repeat as well. They found that there was less reliance on U2AF2 protein than in humans, in which the protein is required for the splicing process to occur. The pattern of repeating base pairs around introns that alters RNA secondary structure

9960-464: The exchanging the hydrogen atoms that are part of the Watson-Crick base pairing by metal ions. Introduction of metal ions into a DNA duplex has shown to have potential magnetic or conducting properties, as well as increased stability. Metal complexing has been shown to occur between natural nucleobases . A well-documented example is the formation of T-Hg-T, which involves two deprotonated thymine nucleobases that are brought together by Hg and forms

10080-503: The fields of developmental biology , oncology , toxicology , reproductive studies, teratology , genetics , neurobiology , environmental sciences , stem cell research, regenerative medicine , muscular dystrophies and evolutionary theory . As a model biological system, the zebrafish possesses numerous advantages for scientists. Its genome has been fully sequenced , and it has well-understood, easily observable and testable developmental behaviors. Its embryonic development

10200-443: The first few cell divisions. Fertilized eggs almost immediately become transparent, a characteristic that makes D. rerio a convenient research model species . Sex determination of common laboratory strains was shown to be a complex genetic trait, rather than to follow a simple ZW or XY system. The zebrafish embryo develops rapidly, with precursors to all major organs appearing within 36 hours of fertilization. The embryo begins as

10320-476: The first few days (72 h panel). After a few months, the adult fish reaches reproductive maturity (bottom panel). To encourage the fish to spawn, some researchers use a fish tank with a sliding bottom insert, which reduces the depth of the pool to simulate the shore of a river. Zebrafish spawn best in the morning due to their Circadian rhythms . Researchers have been able to collect 10,000 embryos in 10 minutes using this method. In particular, one pair of adult fish

10440-409: The first species to be sold as GloFish (fluorescent colored fish). In late 2003, transgenic zebrafish that express green , red, and yellow fluorescent proteins became commercially available in the United States. The fluorescent strains are trade-named GloFish ; other cultivated varieties include "golden", "sandy", "longfin" and "leopard". The leopard danio, previously known as Danio frankei ,

10560-474: The function of the protein moiety encoded by that exon or can sometimes knock down the protein activity altogether. These molecules have been applied to studies in several model organisms , including mice , zebrafish , frogs and sea urchins . Morpholinos can also modify the splicing of pre-mRNA or inhibit the maturation and activity of miRNA. Techniques for targeting Morpholinos to RNAs and delivering Morpholinos into cells have recently been reviewed in

10680-626: The heavy strand. In between a grouping of five tRNA genes, a sequence resembling vertebrate origin of light strand replication is found. It is difficult to draw evolutionary conclusions because it is difficult to determine whether base pair changes have adaptive significance via comparisons with other vertebrates' nucleotide sequences. T-boxes and homeoboxes are vital in Danio similarly to other vertebrates. The Bruce et al. team are known for this area, and in Bruce et al. 2003 & Bruce et al. 2005 uncover

10800-1013: The mature mRNA. Targeting some splice targets results in intron inclusions, while activation of cryptic splice sites can lead to partial inclusions or exclusions. Targets of U11 / U12 snRNPs can also be blocked. Splice modification can be conveniently assayed by reverse-transcriptase polymerase chain reaction ( RT-PCR ) and is seen as a band shift after gel electrophoresis of RT-PCR products. Morpholinos have been used to block miRNA activity and maturation. Fluorescein -tagged Morpholinos combined with fluorescein-specific antibodies can be used as probes for in-situ hybridization to miRNAs. Morpholinos can block ribozyme activity. U2 and U12 snRNP functions have been inhibited by Morpholinos. Morpholinos targeted to "slippery" mRNA sequences within protein coding regions can induce translational frameshifts . Morpholinos can block RNA editing, poly(A) tailing and translocation sequences. Morpholino activities against this variety of targets suggest that Morpholinos can be used as

10920-435: The microenvironment has been utilized in studies of e.g. structure and dynamics within both DNA and RNA, dynamics and kinetics of DNA-protein interaction and electron transfer within DNA. A newly developed and very interesting group of fluorescent base analogues that has a fluorescence quantum yield that is nearly insensitive to their immediate surroundings is the tricyclic cytosine family. 1,3-Diaza-2-oxophenothiazine, tC, has

11040-694: The most common aquaculture species. Because the most common are salmonids and cod in the Protacanthopterygii and sea bass , sea bream , tilapia , and flatfish , in the Percomorpha , zebrafish results may not be perfectly applicable. Various other models – Goldfish ( Carassius auratus ), Medaka ( Oryzias latipes ), Stickleback ( Gasterosteus aculeatus ), Roach ( Rutilus rutilus ), Pufferfish ( Takifugu rubripes ), Swordtail ( Xiphophorus hellerii ) – are less used normally but would be closer to particular target species. The only exception are

11160-460: The mother – will prevent initiation of the mesoderm or endoderm development processes in this species.) In 1999, the nacre mutation was identified in the zebrafish ortholog of the mammalian MITF transcription factor. Mutations in human MITF result in eye defects and loss of pigment, a type of Waardenburg Syndrome . In December 2005, a study of the golden strain identified the gene responsible for its unusual pigmentation as SLC24A5 ,

11280-467: The normal exploratory behavior. This demonstrates relationships between glucocorticoids, fluoxetine, and exploration in this fish. Zebrafish have been used as a model for studying DNA repair pathways. Embryos of externally fertilized fish species, such as zebrafish during their development, are directly exposed to environmental conditions such as pollutants and reactive oxygen species that may cause damage to their DNA . To cope with such DNA damages,

11400-404: The oligo is covalently linked to a delivery dendrimer , enter cells when administered systemically in adult animals or in tissue cultures. In eukaryotic organisms, pre-mRNA is transcribed in the nucleus, introns are spliced out, then the mature mRNA is exported from the nucleus to the cytoplasm . The small subunit of the ribosome usually starts by binding at the 5' end of the mRNA and

11520-428: The opportunity, adults eat hatchlings, which may be protected by separating the two groups with a net, breeding box or separate tank. In captivity, zebrafish live approximately forty-two months. Some captive zebrafish can develop a curved spine. They can range from a few centimeters to a few inches, and provide movement in a freshwater fish tank. The zebra danio was also used to make genetically modified fish and were

11640-719: The peptide conjugates, they have been used in vivo for effective oligo delivery at doses below those causing observed toxicity. An octa-guanidinium dendrimer attached to the end of a Morpholino can deliver the modified oligo (called a Vivo-Morpholino) from the blood to the cytosol. Delivery-enabled Morpholinos, such as peptide conjugates and Vivo-Morpholinos, show promise as therapeutics for viral and genetic diseases. Nucleic acid analogues Nucleic acid analogues are compounds which are analogous (structurally similar) to naturally occurring RNA and DNA , used in medicine and in molecular biology research. Nucleic acids are chains of nucleotides, which are composed of three parts:

11760-443: The phosphate linked 3' hydroxy group, making RNA too unstable to be used or synthesized reliably. To overcome this, a ribose analogue can be used. The most common RNA analogues are 2'-O-methyl-substituted RNA, locked nucleic acid (LNA) or bridged nucleic acid (BNA), morpholino , and peptide nucleic acid ( PNA ). Although these oligonucleotides have a different backbone sugar—or, in the case of PNA, an amino acid residue in place of

11880-429: The process, such as Wnt signaling and Fibroblast growth factor . In probing disorders of the nervous system, including neurodegenerative diseases, movement disorders, psychiatric disorders and deafness, researchers are using the zebrafish to understand how the genetic defects underlying these conditions cause functional abnormalities in the human brain, spinal cord and sensory organs. Researchers have also studied

12000-417: The processes of organogenesis and differentiation are past, with observed phenotypes consistent with target-gene knockdown. Control oligos with irrelevant sequences usually produce no change in embryonic phenotype, evidence of the Morpholino oligo's sequence-specificity and lack of non-antisense effects. The dose required for a knockdown can be reduced by coinjection of several Morpholino oligos targeting

12120-468: The product of a redesign of natural nucleic acid structure. Usually 25 bases in length, they bind to complementary sequences of RNA or single-stranded DNA by standard nucleic acid base-pairing . In terms of structure, the difference between Morpholinos and DNA is that, while Morpholinos have standard nucleic acid bases, those bases are bound to methylene morpholine rings linked through phosphorodiamidate groups instead of phosphates . The figure compares

12240-492: The properties of the chain (PNA can even form a triple helix ). Nucleic acid analogues are also called xeno nucleic acids and represent one of the main pillars of xenobiology , the design of new-to-nature forms of life based on alternative biochemistries. Artificial nucleic acids include peptide nucleic acids (PNA), Morpholino and locked nucleic acids (LNA), as well as glycol nucleic acids (GNA), threose nucleic acids (TNA) and hexitol nucleic acids (HNA). Each of these

12360-584: The recommended feeding rate decreases to about 6–8% of their BW per day, with a focus on high-quality dry feeds that meet their protein and energy needs. Upon reaching adulthood (over 90 dpf), zebrafish typically require a feeding rate of around 5% of their BW per day. Throughout these stages, it is essential to adjust the particle size of the feed: less than 100 μm for newly hatched larvae, 100–200 μm for those between 16 and 30 dpf, and larger particles for juveniles and adults. This structured approach to feeding not only supports optimal growth and health but also enhances

12480-551: The reliability of experimental outcomes in research settings Zebrafish are hardy fish and considered good for beginner aquarists. Their enduring popularity can be attributed to their playful disposition, as well as their rapid breeding, aesthetics, cheap price and broad availability. They also do well in schools or shoals of six or more, and interact well with other fish species in the aquarium. However, they are susceptible to Oodinium or velvet disease, microsporidia ( Pseudoloma neurophilia ), and Mycobacterium species. Given

12600-688: The rescue RNA makes recovery of the wild-type phenotype impossible. In embryos, Morpholinos can be tested in null mutants to check for unexpected RNA interactions, then used in a wild-type embryo to reveal the acute knockdown phenotype. The knockdown phenotype is often more extreme than the mutant phenotype; in the mutant, effects of losing the null gene can be concealed by genetic compensation. Because of their completely unnatural backbones, Morpholinos are not recognized by cellular proteins. Nucleases do not degrade Morpholinos, nor are they degraded in serum or in cells. Up to 18% of Morpholinos appear to induce nontarget-related phenotypes including cell death in

12720-469: The rescue mRNA contains no target for the Morpholino. The rescue mRNA's coding region encodes the protein of interest. Translation of the rescue mRNA replaces production of the protein that was knocked down by the Morpholino. Since the rescue mRNA would not affect phenotypic changes due to the Morpholino's off-target gene expression modulation, this return to wild-type phenotype is further evidence of Morpholino specificity. In some cases, ectopic expression of

12840-436: The ribose phosphate—they still bind to RNA or DNA according to Watson and Crick pairing while being immune to nuclease activity. They cannot be synthesized enzymatically and can only be obtained synthetically using the phosphoramidite strategy or, for PNA, other methods of peptide synthesis . Dideoxynucleotides are used in sequencing . These nucleoside triphosphates possess a non-canonical sugar, dideoxyribose, which lacks

12960-505: The role of two of these elements in oocytes of this species. By interfering via a dominant nonfunctional allele and a morpholino they find the T-box transcription activator Eomesodermin and its target mtx2 – a transcription factor – are vital to epiboly . (In Bruce et al. 2003 they failed to support the possibility that Eomesodermin behaves like Vegt . Neither they nor anyone else has been able to locate any mutation which – in

13080-471: The same family of cytosine analogues, a FRET-acceptor base analogue, tC nitro , has been developed. Together with tC as a FRET-donor this constitutes the first nucleic acid base analogue FRET-pair ever developed. The tC-family has, for example, been used in studies related to polymerase DNA-binding and DNA-polymerization mechanisms. In a cell, there are several non-canonical bases present: CpG islands in DNA (often methylated), all eukaryotic mRNA (capped with

13200-444: The same mRNA, which is an effective strategy for reducing or eliminating dose-dependent off-target RNA interactions. mRNA rescue experiments can sometimes restore the wild-type phenotype to the embryos and provide evidence for the specificity of a Morpholino. In an mRNA rescue, a Morpholino is co-injected with an mRNA that codes for the morphlino's protein. However, the rescue mRNA has a modified 5'-UTR (untranslated region) so that

13320-571: The same team at the Scripps Research Institute that first introduced two extra nucleobases into bacterial DNA reported having constructed a semi-synthetic E. coli bacteria able to make proteins using such DNA. Its DNA contained six different nucleobases : four canonical and two artificially added, dNaM and dTPT3 (these two form a pair). The bacteria had two corresponding RNA bases included in two new codons, additional tRNAs recognizing these new codons (these tRNAs also contained two new RNA bases within their anticodons) and additional amino acids, enabling

13440-649: The single-cell or few-cell stage; an alternative method for embryonic delivery is electroporation , which can deliver oligos into tissues of later embryonic stages. Common techniques for delivery into cultured cells include the Endo-Porter peptide (which causes the Morpholino to be released from endosomes ), the Special Delivery system (no longer commercially available, used a Morpholino-DNA heteroduplex and an ethoxylated polyethylenimine delivery reagent), electroporation, or scrape loading. Delivery into adult tissues

13560-580: The structures of the two strands depicted there, one of RNA and the other of a Morpholino. Replacement of anionic phosphates with the uncharged phosphorodiamidate groups eliminates ionization in the usual physiological pH range, so Morpholinos in organisms or cells are uncharged molecules. The entire backbone of a Morpholino is made from these modified subunits. Morpholinos do not trigger the degradation of their target RNA molecules, unlike many antisense structural types (e.g., phosphorothioates , siRNA ). Instead, Morpholinos act by "steric blocking", binding to

13680-444: The targeted proteins become undetectable by Western blot . In 2016 a synthetic peptide-conjugated PMO (PPMO) was found to inhibit the expression of New Delhi Metallo-beta-lactamase , an enzyme that many drug-resistant bacteria use to destroy carbapenems. Morpholinos can interfere with pre-mRNA processing steps either by preventing splice-directing small nuclear ribonucleoproteins ( snRNP ) complexes from binding to their targets at

13800-528: The trade name zebra danio (and thus often called a " tropical fish " although it is both tropical and subtropical ). The zebrafish is an important and widely used vertebrate model organism in scientific research, particularly developmental biology , but also gene function, oncology , teratology , and drug development , in particular pre-clinical development . It is also notable for its regenerative abilities, and has been modified by researchers to produce many transgenic strains. The zebrafish

13920-454: The wild with some variations depending on location. Its lifespan in captivity is around two to three years, although in ideal conditions, this may be extended to over five years. In the wild it is typically an annual species. In 2015, a study was published about zebrafishes' capacity for episodic memory . The individuals showed a capacity to remember context with respect to objects, locations and occasions (what, when, where). Episodic memory

14040-456: The zebrafish reference genome revealed over 5 million single nucleotide variations and over 1.6 million insertion deletion variations. The zebrafish reference genome sequence of 1.4GB and over 26,000 protein coding genes was published by Kerstin Howe et al. in 2013. In October 2001, researchers from the University of Oklahoma published D. rerio's complete mitochondrial DNA sequence. Its length

14160-414: The zebrafish still appeared healthy. The unusually cold temperature was at one of the highest known zebrafish locations at 1,576 m (5,171 ft) above sea level, although the species has been recorded to 1,795 m (5,889 ft). The zebrafish is named for the five uniform, pigmented, horizontal, blue stripes on the side of the body, which are reminiscent of a zebra's stripes, and which extend to

14280-473: The zebrafish to gain new insights into the complexities of human musculoskeletal diseases, such as muscular dystrophy . Another focus of zebrafish research is to understand how a gene called Hedgehog , a biological signal that underlies a number of human cancers, controls cell growth. Inbred strains and traditional outbred stocks have not been developed for laboratory zebrafish, and the genetic variability of wild-type lines among institutions may contribute to

14400-463: Was found in other teleosts , but not in tetrapods . This indicates that an evolutionary change in tetrapods may have led to humans relying on the U2AF2 protein for RNA splicing while these genes in zebrafish undergo splicing regardless of the presence of the protein. D. rerio has three transferrins , all of which cluster closely with other vertebrates . When close relatives mate, progeny may exhibit

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