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Microinjection of Zebrafish Embryos to Analyze Gene Function

1,2, 1, 1,2

1Department of Genetics, Harvard Medical School, 2Department of Cardiology, Children’s Hospital Boston

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    Summary

    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.

    Date Published: 3/09/2009, Issue 25; doi: 10.3791/1115

    Cite this Article

    Rosen, J. N., Sweeney, M. F., Mably, J. D. Microinjection of Zebrafish Embryos to Analyze Gene Function. J. Vis. Exp. (25), e1115, doi:10.3791/1115 (2009).

    Abstract

    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.

    Protocol

    Part 1: Egg production and collection

    1. 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.
    2. 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.
    3. 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.
    4. 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)

    Part 2: Needle pulling, loading, and preparation

    1. 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.
    2. 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.
    3. 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.

    Part 3: Injection

    1. Ensure the embryos have not developed past the four-cell stage. Ideally, embryos should be at the one-cell stage.
    2. Lower the needle toward the column of eggs, holding the dish in place with your opposite hand.
    3. 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.
    4. 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.

    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).

    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).

    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.

    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.

    Discussion

    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’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 μM and 500 μM 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.

    Disclosures

    Acknowledgements

    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.

    Materials

    Name Type Company Catalog Number Comments
    Microinjector Tool Harvard Apparatus PLI 100
    Micromanipulator Tool Narishige International MN 153
    Needle Puller Tool Sutter Instrument Co. P 97
    Glass Capillaries Tool World Precision Instruments, Inc. TW100 F6
    Microloader Pipettes Tool Eppendorf 5242956.003
    Needle Holder Tool World Precision Instruments, Inc. MPH310

    References

    1. Mably, J.D., Mohideen, M.P.K., Burns, C.G., Chen, J., & Fishman, M.C. heart of glass regulates the concentric growth of the heart in zebrafish. Curr. Biol. 13, 2138-2147, (2003).

    Comments

    19 Comments

    Request:           Respested sir/ madam                                Please can you send the protocol of DNA isolation, RAPD, and Procedure, culture media composition etc for protoplast culture. thanks.
    Reply

    Posted by: atanu d.April 2, 2009, 5:09 AM

    You must have posted on the wrong protocol.  Ours is for zebrafish embryo microinjection.  Good luck finding the information you need!
    Reply

    Posted by: AnonymousApril 2, 2009, 3:24 PM

    My translation-blocking morpholino is carboxy-fluoresecin tagged but I don't see the fluorescence under the microscope after injection? Can the concentration be too low? How dŒs the tag work? Thank you 
    Reply

    Posted by: AnonymousApril 17, 2009, 10:05 PM

    Because the fluorescence of carboxyfluorescein dŒs not require any morpholino-related mechanism, I would verify that the injection material alone is fluorescing under your microscope. The tag is simply attached to the morpholino during manufacturing to ensure the presence of the morpholino in the injected tissue. The final concentration within the embryo would have to be extremely low to not be visible at all.    
    Reply

    Posted by: Michael S.April 21, 2009, 10:47 AM

    Hey guys do you see any intergration events when injecting into the yolk? Would you expect to see a greater number intergration events if you injected directly into the cell? Have you seen yolk glowers when injecting reporter genes (e.g RXP/GFP) and if so what do you think is occuring here? Intergaration?

    Regards

    Giles
    Reply

    Posted by: AnonymousJune 22, 2009, 3:40 AM

    Hi Giles,
    When you inject DNA into the yolk, you get some integration, but not as much as when you inject into the cell. If you use the tol² system and inject DNA with particular flanking sequences and transposase RNA, you get a huge amount of integration. We do see yolk glowers; we're not sure whether this is due to maintenance of the plasmid as an episome, or whether integration has occured.
    Reply

    Posted by: AnonymousJune 24, 2009, 1:11 PM

    Hi, Jonathan Rosen
    First of all thanks for such an informative presentation. I really liked it a lot and I am highly impressed with the techniques which has been used by your lab.Could you send the protocol for the microinjection of zebrafish embryo to analyse gene function. Also can you please let me know which equipment you use to displace embryos from one place to another inside the petridish or in agarose gel.
    I shall be thankful to you
    Kind Regards
    Avdesh
    chaudharyavdesh@gmail.com
    Reply

    Posted by: AnonymousAugust 23, 2009, 10:18 AM

    dŒs the orientation of the embryo matter prior to injection? i always thought that the needle should pierce opposite to the cell, through the yolk sac. also, can a thick, firm needle damage the embryo, as opposed to a thin unsupported one?
    thanks
    Reply

    Posted by: AnonymousAugust 12, 2010, 9:16 PM

    I find it easiest to inject without going through the cell. Too thick a needle can definitely damage the embryo; if you're seeing a lot of yolk leak out after you inject, the needle is too thick.
    Reply

    Posted by: AnonymousAugust 17, 2010, 6:06 PM

    Sir i would like to know a simple study using wild type embryos of the Zebra fish with out using GFP
    Reply

    Posted by: AnonymousAugust 25, 2010, 2:20 AM

    Sir i would like know a simple invitro study on the wild type embryos of Zebrafish with out GFP
    Reply

    Posted by: AnonymousAugust 25, 2010, 2:22 AM

    Hi, Thanks for this video! Other protocols suggest injecting DNA/RNA/morpholino dissolved in KCl or Danieau's. Do you use one of these or just miliQ water, and do you think the solution makes a difference for survival? Thanks!
    Reply

    Posted by: AnonymousJanuary 24, 2012, 10:21 AM

    Hi. Glad the video was helpful. Although KCl or Danieau's solution are often used, we haven't found that either improves embryo survival over just water. I believe Gene-Tools recommends dissolving the stock morpholino in water now, rather than Danieau's solution as they recommended in the past. They may have done a more thorough comparison of the different solutions and their influence on embryo development.

    Good luck with your studies.
    Reply

    Posted by: AnonymousJanuary 25, 2012, 4:26 PM

    I am trying micro injection of EGFP-N3 vector to standardize the injection protocol. But in all my attempts, embryos are dying. what might be the reason. I am using methylene blue as a tracker. Please help me out.
    Reply

    Posted by: AnonymousFebruary 7, 2012, 7:23 AM

    I have never tried it with methylene blue. I would use phenol red. I have also found that I get more lethality from DNA injection than from RNA injection. If you want to globally over-express a gene early in embryonic development, RNA is better anyway because it will go into every cell, whereas DNA will be mosaic.
    Reply

    Posted by: AnonymousFebruary 7, 2012, 10:55 AM

    I WAS WONDERIN IF U HAVE EXPERIENCE USING TH PARASITE BLASTOCYST HOMINIS AS THE SHELL TO INJECTED OTHER PARASITES INTO AS MEANS OF PROTEIN PRODUCTION IF SO IWAS INTERESTED IN UR FININDINDS TO COMPARE TO THE THE VERY STRANG FINDINS FROM MY PROJECT
    Reply

    Posted by: AnonymousMarch 27, 2012, 8:07 PM

    How to prepare egg water
    Reply

    Posted by: Yumei C.July 22, 2012, 11:25 PM

    http://staff.missouriwestern.edu/users/daggett/PR1D.ZF.E3.pdf
    Reply

    Posted by: Michael S.November 6, 2012, 6:38 PM

    Hello,
    I am currently using a Hsp70I promoter in the Tol² system for generating transgenic zebrafish. Any suggestions for the best time and condition to do heat shock after microinjecton ? Is Hsp70I promoter leaky after recovery from heat shock? Your suggestions and comments will be highly appreciated.
    Reply

    Posted by: Hsu k.March 6, 2013, 3:19 AM

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