We acknowledge members of the Mably lab and Narie Storer for their technical assistance, and the American Heart Association (NCRP Scientist Development Grant 0635363N) and the National Heart, Lung, and Blood Institute (Grant SCCOR RFA HL02-027) for funding.

Part 1: Egg production and collection
<ol>
<li>The night prior to injection, set up the fish in breeding tanks with dividers in place. To increase total egg production, fish can be set up in a ratio of two females to one male if desired.</li>
<li>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.</li>
<li>Using a strainer, collect the eggs from the breeding cages and rinse them with egg water. Pour eggs into a Petri dish with egg water and remove unfertilized eggs and debris with a transfer pipette. Fish can be regrouped in larger tanks to produce additional rounds of eggs for injection. Adjust the timing of egg collection to allow for maximum numbers of eggs to be produced without letting them pass the single cell stage.</li>
<li>Place a microscope slide in the inverted lid of a 100mm Petri dish. Use a transfer pipette to line up the eggs against the side of the slide forming a single column. Remove excess egg water from the slide by pressing a Kimwipe against the side opposite the eggs. (Figure 1A)</li>
</ol>
Part 2: Needle pulling, loading, and preparation
<ol>
<li>With a micropipette puller, pull a 1.0mm OD glass capillary into two needles and store in a 150mm Petri dish by laying over silly putty ramps. Needles can be pulled in advance.</li>
<li>Backload the needle with 3 μL of injection material using a microloader pipette. Shake the bolus toward the needle tip until there are few or no bubbles remaining.</li>
<li>Turn on the air source and microinjector. Insert the needle into the microinjector and insure a tight seal within the housing. Check that the micromanipulator is in a proper position to allow for a wide range of movement and adjustment. Bring the needle tip into the plane of view of the microscope, high off the stage, and focus on the thinnest region of the tip. Use a pair of sharp forceps to pinch off the needle at a point which leaves the needle narrow enough to pierce the chorion and yolk but still capable of delivering a consistent bead size. A drop of mineral oil on a micrometer can be used to calculate the volume of each injection. When injected into the oil, a bead with a diameter of 0.1 mm contains 500 pL of injection material (figure 1B); injection volumes of 500 pL or 1nL are typically used. Depress the foot pedal and monitor the size of the bead while trimming the needle and adjusting the injection pressure as needed. Ideal injection volumes will fill approximately 10% of the egg volume. The quality of the needle tip is crucial to both the ease of injection and the quality and consistency of the results.</li>
</ol>
Part 3: Injection
<ol>
<li>Ensure the embryos have not developed past the four-cell stage. Ideally, embryos should be at the one-cell stage.</li>
<li>Lower the needle toward the column of eggs, holding the dish in place with your opposite hand.</li>
<li>Pierce the surface of the chorion and enter the yolk in one smooth stroke while watching for any crushing or tearing of the yolk sac. Inject the injection material into the yolk (figure 1C). Avoid injecting air bubbles or stretching the yolk as either can be lethal to the embryo. Working down the line, adjust the pressure as needed to maintain a consistent bead size and, using a pipette tip, remove eggs which look unfertilized or are destroyed during the injection process.</li>
<li>After completing a column of eggs, use a gentle stream of egg water to move the injected eggs into a clean Petri dish. Repeat as necessary. Keep several uninjected embryos as a control. At the end of day one, remove dead embryos and record the number of injected embryos. Replace the egg water in the dish periodically to reduce the chance of infection.</li>
</ol>
Part 4: Representative results
Depending on what is being injected, embryos may survive at a lower rate than their uninjected siblings. Fortunately, it is easy to inject huge numbers of them, so this is rarely a problem. It is normal for morphants to exhibit slightly delayed development.
To illustrate the results of microinjection, we used this protocol to manipulate levels of the protein Heart of Glass (Heg). Heg is a transmembrane protein required for normal heart development. In its absence, embryos develop hearts with giant inflow tracts and chambers1. We injected two previously undescribed morpholinos against heg, heg_e3i3_egfr1 and heg_e4i4_egfr2, at a concentration of 500 μM. At 2 days post fertilization, the uninjected sibling controls appear normal, as expected (figure 2A and B). Embryos injected with heg_e3i3_egfr1 have brain edema (figure 2C). Although the hearts of most of these morphants have altered morphology, their chambers are normally sized (figure 2D). Embryos injected with heg_e4i4_egfr2 exhibit variable heart defects. Some appear normal, while others have large inflow tracts and moderately enlarged atria (figure 2E and F). The heg mutant, as previously reported1, has a hugely enlarged inflow tract and atrium (figure 2G and H).
We also injected wildtype embryos with 400 ng/μL mRNA transcribed from full-length heg cDNA. While uninjected controls develop normally (figure 3A and B), embryos injected with heg mRNA exhibit a spectrum of phenotypes ranging from wildtype appearance to severe cyclopia, shortening of the main body axis, necrosis of the head and body, and midline defects (figure 3C and D).
<img src="/files/ftp_upload/1115/Figure%201%20FINAL.png" alt="Embryonic development stages, microscope image, cellular division, early growth analysis." data-altupdatedat="1747911167000">
Figure 1.  Embryos to be injected are lined up against a microscope slide in a Petri dish (A).  Injection volume is determined by injecting into mineral oil placed on a micrometer.  An injection volume of 500 pL, which is commonly used, has a diameter of 0.1 mm (B).  Immediately after injection, the morpholino or mRNA is visible as a punctuate spot in the yolk (C).
<img src="/files/ftp_upload/1115/Figure%202%20FINAL.png" alt="Zebrafish embryonic development stages, panels show morphological changes, microscope image." data-altupdatedat="1747911167000">
Figure 2.  At 2 days post fertilization, uninjected embryos have no gross defects (A) or heart phenotype (B).  Embryos injected with the morpholino heg_e3i3_egfr1 have brain edema (C, arrow) but normally sized heart chambers (D).  Some embryos injected with the morpholino heg_e4i4_egfr2 have enlarged inflow tracts and atria (E,F).  heg mutants have severely enlarged inflow tracts and atria (G,H).  (A),(C),(E), and (G) are 4x DIC images; (B),(D),(F) and (H) are 20x DIC images. a=atrium, i t= inflow tract.
<img src="/files/ftp_upload/1115/Figure%203%20FINAL.png" alt="Zebrafish embryo morphology, microscope images showing developmental stages, comparative analysis." data-altupdatedat="1747911167000">
Figure 3. At 24 hours post fertilization, uninjected embryos have a long, straight body with no necrosis or midline defects (A) and two clearly defined eyes with a neural tube between them (B).  Some embryos injected with heg mRNA, in contrast, exhibit a shortened body axis, necrosis, misshaped somites (C), and cyclopia (D).  (A) and (C) are 4x DIC images; (B) and (D) are 20x DIC images.

<ol>
	<li>Mably, J. D., Mohideen, M. P. K., Burns, C. G., Chen, J., Fishman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=heart+of+glass+regulates+the+concentric+growth+of+the+heart+in+zebrafish.">heart of glass regulates the concentric growth of the heart in zebrafish.</a> Curr. Biol. 13, 2138-2147 (2003).</li></ol>

One of the strengths of the zebrafish model system is the ease with which specific gene products can be added to or eliminated from the embryo by microinjection. To ubiquitously overexpress a particular protein, the mRNA encoding it is injected into the yolk at the 1-cell stage. During the embryo&rsquo;s subsequent development, the RNA is distributed throughout the organism and translated. Conversely, to eliminate a particular protein, morpholinos are used. Morpholinos are synthetic oligonucleotides designed with antisense complementarity to specific RNAs. Like mRNAs, morpholinos are injected into the yolk at the 1-cell stage. Inside the embryo they bind their target RNAs, preventing translation.
The main modification that needs to be made for each morpholino or mRNA is the concentration of injected material. Too high a concentration of either morpholino or mRNA may cause non-specific toxicity; while each morpholino must be tested empirically to determine its optimal concentration, typically morpholino concentrations between 200 &mu;M and 500 &mu;M knock down gene activity effectively without causing non-specific defects. The precise size of the needle opening is not crucial. Within a range of needle sizes, injection pressure and injection time can be adjusted to produce a bolus of the correct volume.
Morpholinos have many applications, including the functional dissection of domains within a protein. When designed to target specific exon-intron junctions, morpholinos will prevent splicing from occurring there. We examined the effects of two morpholinos that bind exon-intron junctions in the pre-mRNA of heg. The morpholino heg_e3i3_egfr1 binds the junction between exon 3 and intron 3, preventing the splicing machinery from incorporating exon 3 into mature transcripts. The morpholino heg_e4i4_egfr2 similarly removes exon 4. Both exons 3 and 4 encode domains containing EGF-like repeats. Some embryos injected with heg_e4i4_egfr2 moderately phenocopy the mutant; further experiments will be required to understand the role of Heg exon 4 in heart development. Surprisingly, embryos injected with heg_e3i3_egfr1 have brain edema, a feature not seen in heg mutants. This may be due to the ability of morpholinos to bind maternal transcripts that would be unaffected in zygotic mutants.

<table><tbody><tr><td>Microinjector</td><td>Tool</td><td>Harvard Apparatus</td><td>PLI 100</td><td></td></tr><tr><td>Micromanipulator</td><td>Tool</td><td>Narishige International</td><td>MN 153</td><td></td></tr><tr><td>Needle Puller</td><td>Tool</td><td>Sutter Instrument Co.</td><td>P 97</td><td></td></tr><tr><td>Glass Capillaries</td><td>Tool</td><td>World Precision Instruments, Inc.</td><td>TW100 F6</td><td></td></tr><tr><td>Microloader Pipettes</td><td>Tool</td><td>Eppendorf</td><td>5242956.003</td><td></td></tr><tr><td>Needle Holder</td><td>Tool</td><td>World Precision Instruments, Inc.</td><td>MPH310</td><td></td></tr></tbody></table>

microinjection of zebrafish embryos to analyze gene function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

Watch this Scientific Journal Video about Microinjection of Zebrafish Embryos to Analyze Gene Function at JoVE.com

Microinjection of Zebrafish Embryos to Analyze Gene Function

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

microinjection-of-zebrafish-embryos-to-analyze-gene-function

Research

JoVE Journal

Biology

77.4K Views.  Harvard Medical School. Deze video laat zien hoe morfolino of mRNA kan worden geïnjecteerd in zebravis embryo's in het een-cellig stadium te verlagen of te verhogen van het niveau van specifieke gen-producten tijdens de verdere ontwikkeling.

Video: Microinjection of Zebrafish Embryos to Analyze Gene Function

Labeling and Imaging Cells in the Zebrafish Hindbrain

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish embryos, injection of plasmid DNA results in mosaic expression, allowing for the visualization of single cells or small clusters of cells 1 . We describe how injection of plasmid DNA encoding membrane-targeted Green Fluorescent Protein (mGFP) under the control of a ubiquitous promoter can be used for imaging cells undergoing neurulation. Central to this protocol is the methodology for imaging labeled cells at high resolution in sections and also in real time. This protocol entails the injection of mGFP DNA into young zebrafish embryos. Embryos are then processed for vibratome sectioning, antibody labeling and imaging with a confocal microscope. Alternatively, live embryos expressing mGFP can be imaged using time-lapse confocal microscopy. We have previously used this straightforward approach to analyze the cellular behaviors that drive neural tube formation in the hindbrain region of zebrafish embryos 2. The fixed preparations allowed for unprecedented visualization of cell shapes and organization in the neural tube while live imaging complemented this approach enabling a better understanding of the cellular dynamics that take place during neurulation.

Key to understanding the morphogenetic processes that shape the early embryo is the ability to image cells at high resolution. We describe here a technique for labeling single cells or small clusters of cells in whole zebrafish embryos with membrane-targeted Green Fluorescent Protein.

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish ...

Neuroscience

Microbead Implantation in the Zebrafish Embryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic conservation, as well as similarities in cellular, molecular, and physiological processes, with other vertebrates including humans. During early ontogeny, zebrafish embryos are optically transparent, allowing researchers to visualize the dynamics of organogenesis using a simple stereomicroscope. Microbead implantation is a method that enables tissue manipulation through the alteration of factors in local environments. This allows researchers to assay the effects of any number of signaling molecules of interest, such as secreted peptides, at specific spatial and temporal points within the developing embryo. Here, we detail a protocol for how to manipulate and implant beads during early zebrafish development.

The zebrafish is an excellent model system for genetic and developmental studies. Bead implantation is a valuable tissue manipulation technique that can be used to interrogate developmental mechanisms by introducing alterations in local cellular environments. This protocol describes how to perform microbead implantation in the zebrafish embryo.

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

Developmental Biology

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.

Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

Manipulation of gene expression in tissues is required to perform functional studies. In this paper, we demonstrate the cerebroventricular microinjection (CVMI) technique as a means to modulate gene expression in the adult zebrafish brain. By using CVMI, substances can be administered into the cerebroventricular fluid and be thoroughly distributed along the rostrocaudal axis of the brain. We particularly focus on the use of antisense morpholino oligonucleotides, which are potent tools for knocking down gene expression in vivo. In our method, when applied, morpholino molecules are taken up by the cells lining the ventricular surface. These cells include the radial glial cells, which act as neurogenic progenitors. Therefore, knocking down gene expression in the radial glial cells is of utmost importance to analyze the widespread neurogenesis response in zebrafish, and also would provide insight into how vertebrates could sustain adult neurogenesis response. Such an understanding would also help the efforts for clinical applications in human neurodegenerative disorders and central nervous system regeneration. Thus, we present the cerebroventricular microinjection method as a quick and efficient way to alter gene expression and neurogenesis response in the adult zebrafish forebrain. We also provide troubleshooting tips and other useful information on how to carry out the CVMI procedure.

In this article, we demonstrate a method for manipulation of gene expression in the ventricular cells of the adult zebrafish telencephalon using antisense morpholino oligonucleotides. We present this method as an efficient and quick protocol that can be used for functional studies in the adult vertebrate brain.

Manipulation of gene expression in tissues is  required to perform functional studies. In this paper, we demonstrate the  cerebroventricular ...

Design and Microinjection of Morpholino Antisense Oligonucleotides and mRNA into Zebrafish Embryos to Elucidate Specific Gene Function in Heart Development

The morpholino oligomer-based knockdown system has been used to identify the function of various gene products through loss or reduced expression. Morpholinos (MOs) have the advantage in biological stability over DNA oligos because they are not susceptible to enzymatic degradation. For optimal effectiveness, MOs are injected into 1-4 cell stage embryos. The temporal efficacy of knockdown is variable, but MOs are believed to lose their effects due to dilution eventually. Morpholino dilution and injection amount should be closely controlled to minimize the occurrence of off-target effects while maintaining on-target efficacy. Additional complementary tools, such as CRISPR/Cas9 should be performed against the target gene of interest to generate mutant lines and to confirm the morphant phenotype with these lines. This article will demonstrate how to design, prepare, and microinject a translation-blocking morpholino against hand2 into the yolk of 1-4 cell stage zebrafish embryos to knockdown hand2 function and rescue these "morphants" by co-injection of mRNA encoding the corresponding cDNA. Subsequently, the efficacy of the morpholino microinjections is assessed by first verifying the presence of morpholino in the yolk (co-injected with phenol red) and then by phenotypic analysis. Moreover, cardiac functional analysis to test for knockdown efficacy will be discussed. Finally, assessing the effect of morpholino-induced blockage of gene translation via western blotting will be explained.

The present protocol describes designing, preparing, and microinjecting a translational-blocking morpholino against a representative cardiac gene; Heart And Neural Crest Derivatives Expressed2 (hand2) into the yolk of newly fertilized zebrafish embryos to knock down gene function. It also shows a transient rescue of these "morphants" by co-injection of mRNA encoding this gene product.

The morpholino oligomer-based knockdown system has been used to identify the function of various gene products through loss or reduced expression.  ...

Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function. Our studies have found that dying cells send signals to their live neighbors to form and contract a ring of actin and myosin that ejects it out from the epithelial sheet while closing any gaps that might have resulted from its exit, a process termed cell extrusion1. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe a method to induce and image extrusion in the larval zebrafish epidermis. To visualize extrusion, we inject a red fluorescent protein labeled probe for F-actin into one-cell stage transgenic zebrafish embryos expressing green fluorescent protein in the epidermis and induce apoptosis by addition of G418 to larvae. We then use time-lapse imaging on a spinning disc confocal microscope to observe actin dynamics and epithelial cell behaviors during the process of apoptotic cell extrusion. This approach allows us to investigate the extrusion process in live epithelia and will provide an avenue to study disease states caused by the failure to eliminate apoptotic cells.

Dying cells are extruded from epithelial tissues by concerted contraction of neighboring cells without disrupting barrier function. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe methods to induce and image extrusion in the larval zebrafish epidermis at cellular resolution.

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function.  Our studies have found ...

An Efficient Protocol to Assess ERK Activity Modulation in Early Zebrafish Noonan Syndrome Models via Live FRET Microscopy and Immunofluorescence

RASopathies are genetic syndromes caused by ERK hyperactivation and resulting in multisystemic diseases that can also lead to cancer predisposition. Despite a broad genetic heterogeneity, germline gain-of-function mutations in key regulators of the RAS-MAPK pathway underlie the majority of the cases, and, thanks to advanced sequencing techniques, potentially pathogenic variants affecting the RAS-MAPK pathway continue to be identified. Functional validation of the pathogenicity of these variants, essential for accurate diagnosis, requires fast and reliable protocols, preferably in vivo. Given the scarcity of effective treatments in early childhood, such protocols, especially if scalable in cost-effective animal models, can be instrumental in offering a preclinical ground for drug repositioning/repurposing. 
Here we describe step-by-step the protocol for rapid generation of transient RASopathy models in zebrafish embryos and direct inspection of live disease-associated ERK activity changes occurring already during gastrulation through real-time multispectral F&#246;rster resonance energy transfer (FRET) imaging. The protocol uses a transgenic ERK reporter recently established and integrated with the hardware of commercial microscopes. We provide an example application for Noonan syndrome (NS) zebrafish models obtained by expression of the Shp2D61G. We describe a straightforward method that enables registration of ERK signal change in the NS fish model before and after pharmacological signal modulation by available low-dose MEK inhibitors. We detail how to generate, retrieve, and assess ratiometric FRET signals from multispectral acquisitions before and after treatment and how to cross-validate the results via classical immunofluorescence on whole embryos at early stages. We then describe how, via examining standard morphometric parameters, to query late changes in embryo shape, indicative of a resulting impairment of gastrulation, in the same embryos whose ERK activity is assessed by live FRET at 6 h post fertilization.

RASopathies are multisystem genetic syndromes caused by RAS-MAPK pathway hyperactivation. Potentially pathogenic variants awaiting validation emerge continuously while poor preclinical evidence limits therapy. Here, we describe our in vivo protocol to test and cross-validate RASopathy-associated ERK activation levels and its pharmacological modulation during embryogenesis by live FRET imaging in Teen-reporter zebrafish.

RASopathies are genetic syndromes caused by ERK hyperactivation and resulting in multisystemic diseases that can also lead to cancer predisposition. ...

Biochemistry

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

Zebrafish Microinjection Techniques

One of the major advantages to working with zebrafish (Danio rerio) is that their genetics can be easily manipulated by microinjection of early stage embryos. Using this technique, solutions containing genetic material or knockdown constructs are delivered into the blastomeres: the embryonic cells sitting atop the yolk of the newly fertilized egg. Delivery into the cytoplasm is achieved either through direct injection into the blastomere, or via natural cytoplasmic movements that occur after a solution is injected into the yolk. Successful genetic manipulations are usually followed by quantification of embryonic phenotypes in order to elucidate the genetic mechanisms of development.
This video will provide an introduction to carrying out microinjections in zebrafish embryos. The discussion begins with a review of the essential tools for the technique, including the injection apparatus and the microinjector, which controls fluid movement with pressure pulses of air. Next, important preparatory steps are demonstrated, such as the pouring of agar plates to stabilize embryos during injection and calibration of the microinjection apparatus. The injection procedure is then presented along with tips on when and where injections should be performed. Finally, applications of the microinjection technique are discussed, including gene overexpression via mRNA injection, gene silencing by delivery of antisense morpholino oligonucleotides, and the generation of transgenic zebrafish using specially engineered plasmid DNA.

Introduction to Microinjection of Early Zebrafish Embryos

One of the major advantages to working with zebrafish (Danio rerio) is that their genetics can be easily manipulated by microinjection of early stage ...

Microinjection of Zebrafish Embryos to Analyze Gene Function

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