Morpholino antisense oligonucleotides are widely used to modify gene expression by blocking translation of a targeted protein or by modifying premrna splicing 2,3. Concentrations of the working mo are altered to allow for changes in dosages. The zebrafish is a derived member of the genus brachydanio, of the family cyprinidae. Injections of morpholinos, mrna, and intracardiac injection. In summary, a mo should be validated by comparison to a mutant, and if there is a discrepancy, by injection into embryos homozygous for a null allele or an allele lacking the mobinding site. Phenothiourea sensitizes zebrafish cranial neural crest. Obesity is a major cause of type 2 diabetes mellitus t2dm in mammals. Direct delivery of mif morpholinos into the zebrafish otocyst by injection and. Once the injection process is finished, the embryos. The control morpholino at the same dose produced no discernible effects. C the knock down efficiency was analyzed by assaying the enzymatic activity that converts cortisone to 20. Recently, zebrafish has sparked interest in other branches of the biomedical research. Immediately after injection, the morpholino or mrna is visible as a punctuate spot in the.
During caudal fin regeneration, bony rays are the first structure to differentiate in zebrafish fins, providing a useful. They can be easily manipulated genetically through the use of transgenic technology and gene knockdown via. Morpholine was not bound to serum proteins and was excreted 90% unchanged. Gproteincoupled estrogen receptor 1 gper, formerly known as gpr30 is found to be a trophic and protective factor in mediating action of estrogen in adult brain, while its role in developing brain remains.
During injection, a certain substance such as the nucleic acid, protein, or a certain drug is introduced into the embryo, usually at the early stages of development figure 1. One nanoliter of morpholino or mrna was injected into the embryos at the one. Similarly, numerous photoreceptor mutant animal models present phenotypes that resemble retinal degeneration. Microinjection of mrna and morpholino antisense oligonucleotides in zebrafish embryos. Optimal conditions were achieved when embryos, immediately after fertilization, were immersed with dndmovivo at the initial concentration of either 60 or 40. Optimally, the best concentration for a particular mo should be injected at. We have previously established a zebrafish model of dietinduced obesity dio zebrafish by overfeeding artemia. The zebrafish is a tropical fish native to southeast asia. Introduction to investigate the role of a gene during development, overexpression or misexpression of your gene of interest is a fast assay. 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. The following section describes the basic steps of morpholino design, handling, injection, and analysis in zebrafish embryos.
A primer for morpholino use in zebrafish mary ann liebert, inc. For biosecurity purposes, healthy, breedable, identified adult male carriers at least 10 no older than 9 months of age are required for sperm cryopreservation. Zebrafish danio rerio is a fresh water fish that inhabits rivers in india, pakistan and other places in asia. Many labs use danieaus, although this is an extracellular solution. At 120 h postfertilization hpf, the gut epithelium in control wildtype wt siblings has a. Zebrafish embryo holding trays for injection the zebrafish book design. Photo mediated gene activation by using caged mrna in. A crucial factor associated with ibd onset is the presence of elevated levels of the proinflammatory cytokine tumor necrosis factor tnf in the intestine, signified by the use of antitnf. We routinely order 300 nmol of a morpholino oligo specifically designed for a gene of interest from gene tools, llc philomath, or. All images of uninjected control siblings a,c,e,g and wnt8. The zebrafish information network zfin is the database of genetic and genomic data for the zebrafish danio rerio as a model organism. The working concentration depends on the morpholino dose.
A vivo morpholino is a morpholino oligo with a delivery moiety attached, enabling the oligo to enter cells in adult animals. Gene knockdown by morpholinomodified oligonucleotides in. Compared to mammalian models, zebrafish are relatively easy to manipulate genetically, and there are numerous tools available to do so e. We show that the teleost fish, fugu and zebrafish, have two ribeye genes, ribeye a and ribeye b. We typically store loaded pipettes in the same humid chamber used to load them, after sealing the dish with parafilm. Wholemount in situ hybridization revealed that ribeye a is expressed in tissues containing synaptic ribbons, including the pineal gland, inner ear, and retina. Pdf microinjection of mrna and morpholino antisense. It has a sistergroup relationship with danio aesculapii.
Zebrafish gper may be involved in neuronal development. Dna at a concentration of 10 gml transgenesis experi ments to 50. Injection is the most frequently used technique when working with zebrafish. B substantially reduced eye size and generally delayed development are observed by 48 hpf in response to injection of either of the two pax6b morpholinos. Add 100 ul sterile water to make a 3 mm stock solution. Microinjection of mrna and morpholino antisense oligonucleotides. For this reason, the capillary tended to clog after injection into 2030 embryos. Analyses of pancreas development by generation of gfp. To create an animal model that allows observation of exocrine cell differentiation, proliferation, and morphogenesis in living animals, we used the zebrafish elastasea elaa regulatory sequence to develop transgenic zebrafish that display highly specific. Lessons from morpholinobased screening in zebrafish briefings in. The water channel aquaporin1a1 facilitates movement of. Position the needle in a threedimensional coarse manual manipulator before cutting the needle tip, to an. Injection volume is determined by injecting into mineral oil placed on a micrometer. Morpholinos were both injected at a final concentration of 7 ngembryo.
Characterization of zebrafish dysferlin by morpholino knockout. Zebrafish hsp70 is required for embryonic lens formation. The injected embryos were transferred to fishbreeding water and kept in the dark at 28. Morpholinos arrive in a vial at a concentration of 300 nm. Development of a novel zebrafish model for type 2 diabetes. Humans with mutations in photoreceptorrelated genes develop forms of retinal degeneration, such as retinitis pigmentosa, cone dystrophy, or leber congenital amaurosis.
You can inject mrna or dna of the wildtype gene or a constitutively active form of the gene to investigate its gainoffunction phenotype. Read developmental defects in zebrafish for classification of egf pathway inhibitors, toxicology and applied pharmacology on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips. Injections are done with glass needles filled with the material to be delivered. In vivo electroporation of morpholinos into the adult. In zebrafish danio rerio, microinjection of rna, dna, proteins, antisense. Zebrafish danio rerio is a fresh water fish that inhabits rivers in india. I was checking some books and papers related to biostatistics and i found a no. In order to use the zebrafish as a model vertebrate to investigate the developmental toxicity of 2,3,7,8tetrachlorodibenzopdioxin tcdd, it is essential to know whether one or both forms of the zebrafish aryl hydrocarbon receptor ahr, zfahr1 or zfahr2, mediate toxicity.
Histologically, these tumors most closely resemble human malignant peripheral neural sheath tumors mpnsts. Fish homozygous for the p53 m214k mutation develop tumors at 28% incidence by 16. A read is counted each time someone views a publication summary such as the title, abstract, and list of authors, clicks on a figure, or views or downloads the fulltext. The study of zebrafish as a leading model organism for developmental biology is rapidly expanding. Morpholine is a permitted fda in edible coatings for fruit and vegetables. In zebrafish danio rerio, microinjection of rna, dna, proteins. The concentration of 14cmorpholine in the renal cortex was 6. Information on published morpholino sequences that target apoptosisrelated genes can be found in table 16. Mar 03, 2015 inflammatory bowel diseases ibd, including crohns disease and ulcerative colitis, are intestinal disorders of poorly understood origin and are associated with significant morbidity and mortality. A variety of different glofish are currently on the market.
Microinjection of mrna and morpholino antisense oligonucleotides in zebrafish embryos article pdf available in journal of visualized experiments 727 february 2009 with 340 reads. Together, this core data setpenetrance and toxicity per dosage. Here we show that zebrafish golden mutants share these melanosomal changes and that golden encodes a putative cation exchanger slc24a5 nckx5 that localizes to an intracellular membrane, likely the melanosome or its precursor. Alizarin red s in vivo staining exploring optimal concentrations. On the day of the injection, thaw the rna sample, vortex lightly and spin down briefly. In the past two decades, zebrafish has become one of the preferred in vivo model organisms for studying diverse processes like embryogenesis 8, 9, autophagy, behaviour 11, 12 and sleep. In rescue experiments, injection of fulllength, wildtype slc24a5 transcript into homozygous gol b 1 embryos led to the partial restoration of wildtype pigmentation in both. Gproteincoupled estrogen receptor 1 is involved in brain. Injection into zebrafish embryos at the early stages of development. A novel reagent for safe, effective delivery of substances into cells.
Morpholino oligonucleotides can be used to transiently knockdown septin expression in zebrafish larvae see fig. Microinjection of dna, rna and tracer dyes eppendorf. Matrix metalloproteinase is required for zebra fish. Acute exposure to dexamethasone and hydrocortisone resulted in abnormal zebra fish development including craniofacial abnormalities, altered somitogenesis, blood pooling and pericardial and yolk sac edema as well as increased mmp mrna. Apr 23, 2014 by using a synthetic antisense rna called a morpholino we can also silence the production of individual alpha crystallin proteins in zebrafish embryos. At a morpholino concentration of 2 ngembryo, the heart was dysmorphic and showed aberrant looping and a lack of trabeculation figs. Medaka and zebrafish may be injected using an identi cal solution. Developmental defects in zebrafish for classification of.
To determine the role of zfahr2, an antisense morpholino approach was used to knock down translation. Proper staining of skeletal elements in fish by immersion in fluorochrome solutions demands a compromise between concentration, immersion period, survival and rearing conditions. The zebrafish danio rerio is a freshwater fish belonging to the minnow family of the order cypriniformes. Zebrafish morpholino injection protocol prepared with j. Several hsps were subsequently shown to play roles as molecular chaperones in normal intracellular protein folding and targeting events and to be expressed during discrete periods in the development of several embryonic tissues. The popeye domain containing 2 popdc2 gene in zebrafish. Morpholinos in the zebrafish serve as a powerful reverse genetics tool by knocking down gene function. Analysis of nup3 expression in zebrafish embryos by in situ mrna hybridization ish and.
Morpholino injections cause around 10 25 % death in 24 hour embryos depending. The zebrafish danio rerio has emerged as a powerful model to study. The prox1morpholino was used at a concentration of 1mm and consistently produced reproducible phenotypes with an injection volume around 8 nl that manifested in a drastic reduction of prox1 expression and strong fibrillation defects in the slow muscles. Micro injection of bacteria and multicolor fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.
Lighter variations of pigmentation in humans are associated with diminished number, size, and density of melanosomes, the pigmented organelles of melanocytes. Immediately after injection, the morpholino or mrna is visible as a punctuate spot in the yolk c. In its larval stages it is transparent and as it matures to an adult it develops stripes that run along the length of the body and look blue in colour. Phenol red has been used by several researchers 7,11,34, 37 38394041, however, the effects of adding phenol red to the injection material on the survival rate of fish embryos have not. The use of zebrafish danio rerio as a model organism began in the 1960s. Vivomorpholinos can be used for iv, ip, or local injections, icv infusions, explant cultures and cell cultures. Morpholine is used because its volatility is about the same as water, so once it is added to the water.
In contrast to what we know on development of endocrine pancreas, the formation of exocrine pancreas remains poorly understood. An injection volume of 500 pl, which is commonly used, has a diameter of 0. Mutations in the gene encoding trpm7 trpm7, a member of the transient receptor potential trp superfamily of cation channels that possesses an enzymatically active kinase at its c terminus, cause the touchunresponsive zebrafish mutant touchdown. This group bathed zebrafish embryos with chorion intact in vivomorpholino solutions, with oligos targeting deadend dnd.
The main antisense chemistry used in zebrafish are morpholino phosphorodiamidate oligonucleotides. The ab strain of zebra fish danio rerio, obtained from the zebrafish international resource center zirc, was used for all the experiments described. Knockdown screens searching for novel genes and pathways have been. Zebrafish express both cb1 and cb2 receptors along with. The zebrafish danio rerio is a freshwater fish belonging to the minnow family cyprinidae of the order cypriniformes. The zebrafish danio rerio has long been used as a model for developmental biology, making it an excellent model to use also in developmental toxicology. Injection of volumes higher than 12 nl should be avoided as this can disrupt embryo development. Let agar chamber plates solidify before use and store at 4. Zebrafish as a highthroughput in vivo model for testing. Ribeye is the only known protein specific to synaptic ribbon, but its function is unclear. This study used the zebrafish danio rerio as a vertebrate model of.
Matrix metalloproteinase is required for zebra fish danio. Embryos to be injected are lined up against a microscope slide in a petri dish a. Human and zebrafish synthetic mrna injection were similarly effective at rescue of gsk3. Etiology and functional validation of gastrointestinal. Native to south asia, it is a popular aquarium fish, frequently sold under the trade name zebra danio and thus often called a tropical fish although both tropical and subtropical. Morpholino tips, chien lab protocols zfin community wiki. The glofish is a patented and trademarked brand of genetically engineered fluorescent fish. While the general breeding and handling of zebrafish are detailed in the zebrafish book 15, this. Subfunctionalization of duplicated zebrafish pax6 genes by. Also make sure that your morpholino concentration is not too high as this would be.
For submission of 50 lines or fewer, the strains are provided to the resource center as adult fish only the zirc does not accept embryos. Epigenetic control of intestinal barrier function and. Only embryos injected with a morpholino targeted to slc24a5 either of two splicejunction morpholinos or one start codon morpholino successfully phenocopied golden. Zebrafish course, mullins, august, 2008 microinjection techniques. Zebrafish photoreceptor degeneration and regeneration. As has been observed in other morpholino experiments there were both live normal, as well as dead fish, observed following morpholino injections.
Morpholino antisense oligonucleotides and mechanisms of gene knockdown. Here we describe the application of mos in zebrafish danio rerio for in vivo functional. Myocyte shape appeared to be unaffected, however the development of the myofibrillar apparatus in the popdc2 morphants was less advanced when compared to the control morphant figs. Aliquot the morpholino into multiple paraffinsealed microcentrifuge tubes. Zebrafish represent an established vertebrate model system that helps to bridge the research gap between cell lineinvertebrate studies and mammalian systems. M followed by a lower serially diluted concentration. We usually recovered the residual solution from the microcapillary by using the microloader and reloaded the solution into a new capillary for continuous injection. Trpm7 is required within zebrafish sensory neurons for the.
How to calculate the concentration of morpholinos for microinjection in zebrafish embryos. Zebra fish were maintained and bred in an aquatic habitat recirculation system according to a husbandry protocol approved by the rutgers university animal care and facilities committee. You can inject mrna or dna of the wildtype gene or a. Zebrafish retina manifests anatomical organization and development remarkably conserved in humans, making these fish. Welcome to the morpholino database morpholinos are a powerful knockdown tool that have been proven to efficiently and specifically reduce translation of a target mrna summerton, 1999. A golden fish reveals pigmentation loss in europeans. Zeigler zebrafish diet, 1lb bag during this covid19 pandemic, pentair global operations and supply teams are working diligently to help ensure our valued customers are getting the best possible service and delivery during this time. Microinjection of zebrafish embryos to analyze gene function. How to calculate the concentration of morpholinos for.
Can anyone tell me the correct statistics for zebrafish embryo. One nanoliter of morpholino or mrna was injected into the embryos at the onecell stage. A common, simpler alternative is 1% phenol red, which has the added advantage of letting you track which embryos have been injected. The concentration of a dissolved mo can be calculated by measuring its absorbance at 265 nm in 0. Bathing zebrafish embryos with vivomorpholinos gene tools, llc. Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as salmonella typhimurium and mycobacterium marinum. Gper morpholino knockdown reduced expression in neuron markers. Morpholine is a food contaminant arising from its use as a boiler water additive morpholine is a common additive, in ppm concentrations, for ph adjustment in both fossil fuel and nuclear power plant steam systems. Here we describe the application of mos in zebrafish danio rerio for in vivo. Microinjection of mir142a3p morpholino mo in zebrafish embryos in. For single morpholino injections, the concentration was 0. Morpholino knockdown studies showed that mmp expression is necessary for normal zebra fish embryogenesis. While the initial testing of tetrahydrocannabinol thc using zebrafish occurred in 1975, zebrafish are currently a burgeoning model for testing the bioactivity of cannabinoids.
Determine the morpholino concentration by diluting 5. Morpholino antisense oligonucleotides are highly stable angerer and angerer, chapter 28. Its molecular structure contains dna bases attached to a backbone of methylenemorpholine rings linked through phosphorodiamidate groups. The fibrodysplasia ossificans progressiva r206h acvr1. Injection of the grna into the zygote efficiently mutated the gene as. Determining the concentration of a morpholino stock solution. We inject small quantities of each morpholino into zebrafish embryos when they contain only one to four cells seen at the right with a red tracer dye in an injected embryo using a picoinjector. The present study tested the hypothesis that zebrafish danio rerio aquaporin1a1 aqp1a1 serves as a multifunctional channel for the transfer of the small gaseous molecules, co2 and ammonia, as well as water, across biological membranes. Mar 19, 2009 embryos were injected at the onecell stage with 3 nanoliters in 1. The respective morpholino concentration used is denoted. We identified and characterized a new allele of touchdown, as well as two previously reported alleles, and found that all three alleles. Before injection, dilute stocks to a working concentration in injection buffer. The many advantages of zebrafish include their small size, prolific spawning, rapid development, and transparent embryos.
C morpholino 1 and the standard control morpholino were injected at a concentration of 2 mm, the tenascin. Currently, most protocols used for vital staining of bone rely on alizarin compounds. Zebrafish embryos were microinjected with a translationblocking morpholino oligonucleotide targeted to aqp1a1. For microinjection of morpholino oligonucleotides, first pull needles from borosilicate glass microcapillaries as described previously benard et al. Gene knockdown by morpholinomodified oligonucleotides in the. Zebrafish were the first glofish available in pet stores, and are now sold in bright red, green, orangeyellow, blue, pink, and purple fluorescent colors. Zebrafish granulocyte colonystimulating factor receptor. Injection of 6 ng of mo1, mo2 or mo3 resulted in approximately 37. Control morpholinos and glo1 were diluted to 3 gl with 1. Zfin provides a wide array of expertly curated, organized and crossreferenced zebrafish research data.284 8 523 704 639 1005 237 159 1093 588 1504 295 721 1054 271 1470 398 436 204 896 1427 915 1341 1444 143 945 272 650 755 390 1150 213 270 987 972 404 1134 735 1348 684 10 499 624 1218 586 1260 1194 443 486 1055