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 &mu;L 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 &mu;M. 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/&mu;L 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="" />
Figure 1.&nbsp; Embryos to be injected are lined up against a microscope slide in a Petri dish (A).&nbsp; Injection volume is determined by injecting into mineral oil placed on a micrometer.&nbsp; An injection volume of 500 pL, which is commonly used, has a diameter of 0.1 mm (B).&nbsp; 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="" />
Figure 2.&nbsp; At 2 days post fertilization, uninjected embryos have no gross defects (A) or heart phenotype (B).&nbsp; Embryos injected with the morpholino heg_e3i3_egfr1 have brain edema (C, arrow) but normally sized heart chambers (D).&nbsp; Some embryos injected with the morpholino heg_e4i4_egfr2 have enlarged inflow tracts and atria (E,F).&nbsp; heg mutants have severely enlarged inflow tracts and atria (G,H).&nbsp; (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="" />
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).&nbsp; Some embryos injected with heg mRNA, in contrast, exhibit a shortened body axis, necrosis, misshaped somites (C), and cyclopia (D).&nbsp; (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.

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<td>Microinjector</td>
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<td>Harvard Apparatus</td>
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<td>Tool</td>
<td>Sutter Instruments</td>
<td>P 97</td>
<td>&nbsp;</td>
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<td>World Precision Instruments</td>
<td>TW100 F6</td>
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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

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JoVE Journal

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77.1K Views.  Harvard Medical School. 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.

Video: Microinjection of Zebrafish Embryos to Analyze Gene Function

Microinjection of A. aegypti Embryos to Obtain Transgenic Mosquitoes

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever.  The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever. The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue ...

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 ...

Generating Chimeric Zebrafish Embryos by Transplantation

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

Microinjection of Medaka Embryos for use as a Model Genetic Organism

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1, as in zebrafish and the mouse. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions.
To take advantage of medaka fish whose embryos are transparent and develop externally, microinjection is an essential technique to inject cell-tracers for labeling cells, mRNAs or anti-sense oligonucleotides for over-expressing and knocking-down genes of interest, and DNAs for making transgenic lines.

Medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions. This protocol highlights the key points for successful microinjection into medaka embryos, an important technique for embryological and genetic analysis using medaka and zebrafish in a laboratory.

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows ...

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

Gene Trapping Using Gal4 in Zebrafish

Large clutch size and external development of optically transparent embryos make zebrafish an exceptional vertebrate model system for in vivo insertional mutagenesis using fluorescent reporters to tag expression of mutated genes. Several laboratories have constructed and tested enhancer- and gene-trap vectors in zebrafish, using fluorescent proteins, Gal4- and lexA- based transcriptional activators as reporters 1-7. These vectors had two potential drawbacks: suboptimal stringency (e.g. lack of ability to differentiate between enhancer- and gene-trap events) and low mutagenicity (e.g. integrations into genes rarely produced null alleles). Gene Breaking Transposon (GBTs) were developed to address these drawbacks 8-10. We have modified one of the first GBT vectors, GBT-R15, for use with Gal4-VP16 as the primary gene trap reporter and added UAS:eGFP as the secondary reporter for direct detection of gene trap events. Application of Gal4-VP16 as the primary gene trap reporter provides two main advantages. First, it increases sensitivity for genes expressed at low expression levels. Second, it enables researchers to use gene trap lines as Gal4 drivers to direct expression of other transgenes in very specific tissues. This is especially pertinent for genes with non-essential or redundant functions, where gene trap integration may not result in overt phenotypes. The disadvantage of using Gal4-VP16 as the primary gene trap reporter is that genes coding for proteins with N-terminal signal sequences are not amenable to trapping, as the resulting Gal4-VP16 fusion proteins are unlikely to be able to enter the nucleus and activate transcription. Importantly, the use of Gal4-VP16 does not pre-select for nuclear proteins: we recovered gene trap mutations in genes encoding proteins which function in the nucleus, the cytoplasm and the plasma membrane.

This protocol describes the method of gene trap insertional mutagenesis using Gal4-VP16 as the primary reporter and GFP/RFP as secondary reporters in zebrafish. Approximately one in ten high-expressing F0 fish yield gene trap progeny co-expressing GFP and RFP. The screening procedure can be readily scaled to adapt to the size of the laboratory performing the insertional mutagenesis screen.

Large clutch size and external development of optically transparent embryos make zebrafish an exceptional vertebrate model system for in vivo insertional ...

Force Measurement During Contraction to Assess Muscle Function in Zebrafish Larvae

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional measures of muscle health. In this video article, we demonstrate a method to measure force generation during contraction of zebrafish larval trunk muscle. Force measurements are accomplished by placing an anesthetized larva into a chamber filled with a salt solution. The anterior end of the larva is tied to a force transducer and the posterior end of the larva is tied to a length controller. An isometric twitch contraction is elicited by electric field stimulation and the force response is recorded for analysis. Force generation during contraction provides a measure of overall muscle health and specifically provides a measure of muscle function. Although we describe this technique for use with wild-type larvae, this method can be used with genetically modified larvae or with larvae treated with drugs or toxicants, to characterize muscle disease models and evaluate treatments, or to study muscle development, injury, or chemical toxicity.

Force measurements can be used to demonstrate changes in muscle function due to development, injury, disease, treatment or chemical toxicity. In this video, we demonstrate a method to measure force during a maximal contraction of zebrafish larval trunk muscle.

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional ...

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 ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...