Morpholinos were both injected at a final concentration of 7 ngembryo. In particular, injection of morpholinos into zebrafish embryos often results in a characteristic neural death by 22 hpf ekker and larson 2001. The search for the holy grail of regenerative medicine the ability to grow. Morpholino oligonucleotide mo injection is a standard technique for eliminating gene expression in zebrafish embryos. Embryos to be injected are lined up against a microscope slide in a petri dish a. Injections of morpholinos, mrna, and intracardiac injection. By using a synthetic antisense rna called a morpholino we can also silence the production of individual alpha crystallin proteins in zebrafish embryos. It soon invaded most of eastern europe and great britain. Compared to mammalian models, zebrafish are relatively easy to manipulate genetically, and there are numerous tools available to do so e. Zhiyuan gong and his colleagues at the national university of singapore were working with a gene that encodes the green fluorescent protein gfp, originally extracted from a jellyfish, that naturally produced.
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. Prepare a working solution by diluting the morpholino solution in sterile water and a final concentration of 0. Antisense morpholino treatment to increase smn levels and. I added 150ul of sterile water to make 2 mm stock solution.
The following morning, after the room lights turn on, pull the dividers from several tanks and allow for approximately 20 minutes of undisturbed mating time. Once the injection process is finished, the embryos. Proceed to part 4 when ready to inject this sample. Spinal muscular atrophy sma is caused by deletion or mutation of smn1 and retention of smn2, this in turn leads to reduced levels of the full smn protein. These data indicate that a morpholino can block the activity of a mature mirna duplex in a functional assay. How to check whether micro injection of morpholino in zebra fish is working or not when we dont have the antibody for the specific protein to verify through the western blot. Optimal conditions were achieved when embryos, immediately after fertilization, were immersed with dndmovivo at the initial concentration of either 60 or 40. The original zebrafish or zebra danio, danio rerio is a native of rivers in india and bangladesh. One nanoliter of morpholino or mrna was injected into the embryos at the one.
Zebrafishs growing impact on medical research sciencedaily. Currently, mutations in 19 ribosomal protein genes have been identified in patients. Zebrafish embryo holding trays for injection the zebrafish book design. Injection of 6 ng of mo1, mo2 or mo3 resulted in approximately 37. Morpholinos in the zebrafish serve as a powerful reverse genetics tool by knocking down gene function. Bathing zebrafish embryos with vivomorpholinos gene tools, llc. 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. Injection into zebrafish embryos at the early stages of development. Morpholino oligonucleotides should be resuspended at a concentration of mm in cell culture grade distilled water depcfree. Keap1 also exists in zebrafish which interacts with nrf2 as a negative regulator. The introduction and rapid spread of the zebra mussel in north american waters has caused great concern among industrial and recreational users of these waters.
While the general breeding and handling of zebrafish are detailed in the zebrafish book 15, this. Optimally, the best concentration for a particular mo should be injected at. M followed by a lower serially diluted concentration. Transient knockdown and overexpression reveal a developmental. In 2000 morpholinos where shown to be effective in zebrafish and rapidly.
The zebrafish danio rerio is a freshwater fish belonging to the minnow family of the order cypriniformes. This group bathed zebrafish embryos with chorion intact in vivomorpholino solutions, with oligos targeting deadend dnd. In vivo electroporation of morpholinos into the adult. Zebrafish course, mullins, august, 2008 microinjection techniques. This technology provides a way of rapidly determining gene function in a vertebrate organism and can be accomplished in a matter of weeks. 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 zebra mussel is native to the caspian sea region of russia, but during the late 1700s and early 1800s, newly constructed canals greatly increased the zebra mussels range. Zebra fish microinjection instruments for zebrafish research oocytes, embryos, larvae and adults world precision instruments wpi offers a broad range of research instruments for zebrafish danio rerio research, from the time the genetics of the subject are modified through the point where the physiological effects of the change are monitored. Zebra fish microinjection world precision instruments. As nestin is conserved in zebrafish and its predicted sequence is clustered with the mammalian nestin orthologue, we used zebrafish as a model to investigate its role in embryogenesis.
Morpholino oligos have excellent antisense properties compared to other gene knockdown systems. Zebra mussels threaten fisheries the ultimate bass. High tissue glucose alters intersomitic blood vessels in. Junb controls lymphatic vascular development in zebrafish via. How to check whether micro injection of morpholino in zebra. This paper presents optimising steps towards improving zebra fish embryo production in the laboratory. Zebra fish models for diabetes linkedin slideshare. In zebrafish danio rerio, microinjection of rna, dna, proteins, antisense. 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. Mar 09, 2009 injection volume is determined by injecting into mineral oil placed on a micrometer. Microinjection of zebrafish embryos to analyze gene function. The zebra mussel is believed to have arrived in the u. Determine the morpholino concentration by diluting 5. Constant molecular weight particular to your morpholino x molar.
However, the molecular mechanism of nrf2 regulation in fish has not received due attention 16,17. Zebra mussel dreissena polymorpha ecological risk screening summary u. Immediately after injection, the morpholino or mrna is visible as a punctuate spot in the yolk c. Culture conditions were maintained in the aquaria as stipulated in the oecd. Morpholino oligonucleotides can be used to transiently knockdown septin expression in zebrafish larvae see fig. We typically store loaded pipettes in the same humid chamber used to load them, after sealing the dish with parafilm. Original zebrafish morpholino paper nasevicius a, ekker sc. The importance of p53 in cancer is highlighted by the fact that it is the most frequently mutated tumor suppressor gene in human cancers, with more than half of solid tumors harboring mutations in the p53 locus. The working concentration depends on the morpholino dose. One nanoliter of morpholino or mrna was injected into the embryos at the onecell stage. Aliquot the solution and store at 20c until ready for use. A vivo morpholino is a morpholino oligo with a delivery moiety attached, enabling the oligo to enter cells in adult animals.
Control morpholinos and glo1 were diluted to 3 gl with 1. Morpholino antisense oligonucleotides are highly stable angerer and angerer, chapter 28. Morpholino injections should be performed on 14 cell stage embryos. Research open access microinjection into zebrafish. Native to south asia, it is a popular aquarium fish, frequently sold under the trade name zebra danio and thus often called a tropical. This bivalve mollusk is a biofouler that attaches to any firm substrate e. However, the pathogenic mechanism of dba remains unknown. Immediately after injection, the morpholino or mrna is. 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. Fish and wildlife service, february 2011 revised july 2015. In this demonstration, we will be preparing a working solution of 0. Instead of sealing, we decided to figure 1 survival rates of zebrafish eggs after vehicle injection and 24 h post injection with 4.
Sma is the second leading cause of neuromuscular disease, and many people living with sma need roundtheclock care from their families and also from a variety of medical specialists. 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. T1 lessons from morpholinobased screening in zebrafish. Live data tables purpose day 3 introduction three pertri dishes required for incubating the zebrafish 20 ml of water in each petri dish to allow for the survival of the fish two tubes, one filled with 5ml retonic acid 5ml water and another filled with 10ml retonic acid for the. Native to the drainage basins of the black, caspian and aral seas gollasch and. Position the needle in a threedimensional coarse manual manipulator before cutting the. Gene knockdown by morpholinomodified oligonucleotides in. Zebrafish danio rerio is a fresh water fish that inhabits rivers in india. Improving production of zebra fish embryos in the lab. N2 morpholino oligonucleotides mos are an effective, genespecific antisense knockdown technology used. Many labs use danieaus, although this is an extracellular solution. It measures three centimeters long and has gold and dark blue stripes.
This volume presents a historical perspective on morpholinos, an overview of good morpholino practices, techniques for controlling morpholino activity with light, techniques for modulating microrna activity in zebrafish embryos. I have a morpholino oligo from gene tools typical package size. As has been observed in other morpholino experiments there were both live normal, as well as dead fish, observed following morpholino injections. This phenotype is not specific to the genes targeted for knockdown, as available lossoffunction mutations in corresponding genes do not elicit this neural death. In zebrafish danio rerio, microinjection of rna, dna, proteins.
Nestin is essential for zebrafish brain and eye development. Microinjection of mrna and morpholino antisense oligonucleotides. Morpholino antisense oligonucleotides are widely used to modify gene expression by blocking translation of a targeted protein or by modifying premrna splicing 2,3. Recently, zebrafish has sparked interest in other branches of the biomedical research. Zebrafish danio rerio is a fresh water fish that inhabits rivers in india, pakistan and other places in asia. Vivomorpholinos can be used for iv, ip, or local injections, icv infusions, explant cultures and cell cultures.
Msds files determining the concentration of a morpholino stock solution. However, sealing of any injection hole is a time consuming procedure. May 07, 2009 prepare a working solution by diluting the morpholino solution in sterile water and a final concentration of 0. Coinjection of the mir30c duplex and a morpholino targeting mature mir30c rescues the reporter signal, whereas injection of a control morpholino did not reverse the silencing by mir30c. Morpholino antisense oligonucleotides gene tools, llc, philomath, or are reconstituted as 5 mm stock solutions in sterile distilled water following the manufacturers instructions. Pdf microinjection of mrna and morpholino antisense. Methodologyprincipal findings as nestin is conserved in zebrafish and its predicted sequence is clustered with the mammalian nestin orthologue, we used zebrafish as a model to investigate its role in embryogenesis. We routinely order 300 nmol of a morpholino oligo specifically designed for a gene of interest from gene tools, llc philomath, or. Co injection of the mir30c duplex and a morpholino targeting mature mir30c rescues the reporter signal, whereas injection of a control morpholino did not reverse the silencing by mir30c. A good morpholino experiment starts with oligos that have been carefully designed to minimize offtarget rna binding. Despite its wide use as an npc marker, the function of nestin in embryo development is unclear. Diamondblackfan anemia dba is a rare bone marrow failure syndrome characterized by red blood cell aplasia.
Microinjection of zebrafish embryos to analyze gene. Lossoffunction studies by morpholinomediated knockdown and the crisprcas9 technology identify a novel function for both junb and its target mir182 in lymphatic vascular development in zebrafish. Add 100 ul sterile water to make a 3 mm stock solution. You can inject mrna or dna of the wildtype gene or a. Zebrafish morpholino injection protocol prepared with j. Males are slender and torpedoshaped usually with a pink or yellow tinge. Oct, 2015 lossoffunction studies by morpholino mediated knockdown and the crisprcas9 technology identify a novel function for both junb and its target mir182 in lymphatic vascular development in zebrafish. Characterization of zebrafish dysferlin by morpholino knockout. A primer for morpholino use in zebrafish mary ann liebert, inc. Zebra mussel dreissena polymorpha erss united states fish. They can be easily manipulated genetically through the use of transgenic. Lessons from morpholinobased screening in zebrafish. Direct delivery of mif morpholinos into the zebrafish otocyst by injection and electroporation affects inner ear development.
Effects of zebra mussels on fish population sizes to test their predictions, strayer and his team analyzed data from fish surveys carried out by dec scientists over 26 years, and compared data on population size, growth, and distribution of young fish larvae and juveniles in the river before and after zebra mussels invaded the river in 1991. Jul 19, 2016 zebrafish are becoming more and more popular as a research model for human disease. In many other tumors, p53 is inactivated by more indirect mechanisms, including mdm2 amplification and p14arf deletion. 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 many advantages of zebrafish include their small size, prolific spawning, rapid development, and transparent embryos. How to calculate the concentration of morpholinos for. Zebrafish are becoming more and more popular as a research model for human disease. Gene knockdown by morpholinomodified oligonucleotides in the. Its molecular structure contains dna bases attached to a backbone of methylenemorpholine rings linked through phosphorodiamidate groups. Injection volume is determined by injecting into mineral oil placed on a micrometer.
This knockdown technology is particularly useful in zebrafish as the system is amenable to large scale screening nasevicius and ekker, 2000. The use of zebrafish danio rerio as a model organism began in the 1960s. An injection volume of 500 pl, which is commonly used, has a diameter of 0. Recently, several dba models were generated in zebrafish danio rerio to elucidate the molecular pathogenesis of. On the day of the injection, heat the morpholino solution at 65c for 5 minutes. Background nestin is expressed in neural progenitor cells npc of developing brain. Along with mice and humans, they are one of the most commonly studied animals in biomedical research. Controls, favoring specificity, improving efficacy, storage, and dose.
Morpholinos arrive in a vial at a concentration of 300 nm. For microinjection of morpholino oligonucleotides, first pull needles from borosilicate glass microcapillaries as described previously benard et al. A common, simpler alternative is 1% phenol red, which has the added advantage of letting you track which embryos have been injected. The hepatopancreatic duct cells connect liver hepatocytes and pancreatic acinar cells to the intestine, but the mechanism for their lineage specification is unclear. The night prior to injection, set up the fish in breeding tanks with dividers in place.
How to check whether micro injection of morpholino in. Microinjection or electroporation of morpholino oligos into the embryos of frogs, zebrafish, chicks, sea urchins and other organisms successfully and specifically shuts down the expression of targeted genes, making morpholinos an indispensable tool of developmental. 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. Research open access microinjection into zebrafish embryos. The zebrafish is a tropical fish native to southeast asia. 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. Injection of nestin morpholino mo into fertilized eggs induced time and dosedependent brain and eye developmental defects. Bathing zebrafish embryos with vivomorpholinos gene. Zebrafish embryo holding trays for injectionthe zebrafish book design. To increase total egg production, fish can be set up in a ratio of two females to one male if desired. Four millimolar injection solutions are prepared by combining 20.
Before injection, dilute stocks to a working concentration in injection buffer. 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. Junb controls lymphatic vascular development in zebrafish. Calculate the constant for your morpholino using the following equation. Morpholino antisense oligonucleotides and mechanisms of gene knockdown. Morpholino tips, chien lab protocols zfin community wiki. Introduction to investigate the role of a gene during development, overexpression or misexpression of your gene of interest is a fast assay. The utilization of fish embryos in toxicity testing of hazardous chemicals has recently been adopted in order to satisfy stricter rules and regulations related to using adult animals in toxicity testing. Performing a successful, reproducible, and wellcontrolled morpholino experiment requires oligos that are single stranded and in solution at a known concentration. Animal models shared resource amsr zebrafish science. The zebrafish danio rerio is a freshwater fish belonging to the minnow family cyprinidae of the order cypriniformes. Morpholino oligomers methods and protocols hong m moulton. This study used the zebrafish danio rerio as a vertebrate model of.
366 311 1035 1141 154 230 1525 1217 766 1430 273 1155 1451 294 1239 556 648 949 1115 1097 839 434 45 1338 522 124 975 910 225