Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.


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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.

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Yuan, S., Sun, Z. Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.. J. Vis. Exp. (27), e1113, doi:10.3791/1113 (2009).


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.


Part 1: Preparation of micropipettes, and microinjection chamber plates

  1. Fabricate micropipettes by heating and pulling borosilicate glass capillary tubes (World Precision Instruments, Inc., 1B100-4) in a micropipette puller device (Sutter Instruments Inc., Flaming/Brown P-97). Store in a Petri dish on top of small amount of clay or adhesive tape.
  2. Pour 1.5% agarose (American Bioanlytical, Inc., AB00972-00500) in 1x E3 medium plates molded with wedge-shaped troughs that will serve as an easy method for holding the embryos during the injection (as described in 'The Zebrafish Book") 1. Let agar chamber plates solidify before use and store at 4ºC.

Part 2: Preparation of RNA

One powerful approach for gene function studies is to microinject in vitro transcribed capped RNA in zebrafish embryos. Capped RNA behaves similarly to eukaryotic mRNAs found in vivo due to the presence of the CAP analog. Zebrafish researchers routinely utilize this method to overexpress or misexpress their gene of interest. In this demonstration, we will microinject an EGFP-tagged transcript and use live whole-embryo GFP-expression as a visible readout for a successful injection.

  1. Perform an in vitro CAP RNA transcription reaction on your transcript of interest. In our lab, we routinely insert a cDNA of interest into a pCS2+ vector and perform an in vitro synthesis reaction of large amounts of capped RNA using the mMessage mMachine SP6 kit (Ambion, Inc., AM1340).
  2. Purify the RNA sample by running it through the RNeasy Mini kit (Qiagen, Inc., 74104) or by phenol:chloroform extraction and isopropanol precipitation.
  3. Carefully determine the concentration of the RNA preparation and store at -80ºC until ready for use.
  4. On the day of the injection, thaw the RNA sample, vortex lightly and spin down briefly.
  5. Prepare a working solution with sterile water and a final concentration of 0.025 - 0.050% phenol red (Sigma-Aldrich Co., P0290). Phenol red serves as a visible marker for the injection of the solution into the embryo. In this article, we will prepare a working solution of 100 ng/uL EGFP mRNA with 0.050% phenol red.
  6. Keep working sample on ice and return RNA stock to -80°C.
  7. Proceed to Part 4 when ready to inject this sample.

Part 3: Preparation of morpholino

Morpholino antisense oligonucleotides are widely used to modify gene expression by blocking translation of a targeted protein or by modifying pre-mRNA splicing 2,3. Morpholinos in the zebrafish serve as a powerful reverse genetics tool by knocking down gene function. In this article, we will microinject a morpholino oligo targeted to the translational initiation site of pkd2 (5′-AGGACGAACGCGACTGGAGCTCATC-3′). Based on previously established work, we expect the injected embryos to phenotypically mimic pkd2 mutant fish4.

  1. We routinely order 300 nmol of a morpholino oligo specifically designed for a gene of interest from Gene Tools, LLC (Philomath, OR).
  2. Add 100 uL sterile water to make a 3 mM stock solution. Aliquot the solution and store at -20°C until ready for use.
  3. On the day of the injection, heat the morpholino solution at 65°C for 5 minutes. Snap cool on ice immediately and spin briefly. This step denatures any secondary structures in the oligo and ensures that the solution is completely solubilized.
  4. Prepare a working solution by diluting the morpholino solution in sterile water and a final concentration of 0.025 - 0.050% phenol red. In this demonstration, we will be preparing a working solution of 0.50 mM pkd2 morpholino with 0.050% phenol red.
  5. Keep working solution at room temperature.
  6. Proceed to Part 4 when ready to inject this sample.----

Part 4: Filling the micropipette with your working solution

  1. Cut the distal tip of a micropipette with forceps or a surgical razorblade to create an opening that is just visible under a dissecting microscope (Nikon Instruments, Inc., SM2645) at 50X magnification.
  2. Place 2 uL of your working solution as a drop onto a coverslip.
  3. Attach the back of a micropipette to a 5 mL syringe fitted with tubing on a hypodermic needle and slightly submerge the distal tip of the micropipette into your drop of working solution. Take care not to break the tip of the micropipette.
  4. Siphon the working solution through the distal end of the micropipette by pulling the plunger of the syringe.

Part 5: Calibrating the micropipette injection volume

  1. Place a drop of mineral oil (American Bioanalytical, Inc., AB00921-00025) on a micrometer slide (Fisher, 12-561-SM1).
  2. Turn on power and air supply to the pressure-pulsed micro injector apparatus (World Precision Instruments Inc., PV830).
  3. Attach the micropipette to the micropipette holder of the micro injector apparatus. The micro injector is pressure regulated and discharges are activated by pressing the foot pedal.
  4. Under the dissection microscope, test the injection volume by using the micro injector to place a drop of your working solution onto the micrometer oil. Measure the diameter of the drop as it floats as a sphere on top of the oil.
  5. Adjust the duration and pressure of injection to carefully calibrate your volume of injection. This step aids in the reproducibility of the microinjection experiment. In this demonstration, all microinjections will be done with a drop diameter of 0.15 mm (approximately 1.76 nL in volume).
  6. Once your injection volume has been calibrated, use the “Hold” knob on the micro injector to prevent backfilling and leaking of the micropipette.

Part 6: Preparing fertilized zebrafish embryos for microinjection

  1. Zebrafish will randomly mate in the first few hours of each morning. Collect embryos at the 1-cell stage and place them in 1x E3 medium.
  2. Using a 3 mL transfer pipet (Becton Dickinson Labware, 357524), arrange the embryos along the wedge-shaped troughs in the microinjection chamber plates.
  3. Remove the medium so that the embryos are shallowly submerged and not flooded. This step aids in settling the embryos to the bottom of the troughs as well as in the penetration of the chorion by the micropipette.

Part 7: Microinjection through the chorion

  1. Manipulate the embryos with the micropipette so that the cytoplasm of the 1-cell stage embryo is visible under the dissecting microscope. Take care not to break the tip of the micropipette.
  2. Penetrate the chorion and then the yolk with the micropipette in order to inject into the embryo. In this demonstration, we will be first microinjecting into the yolk directly below the cell, and allow cytoplasmic flow and diffusion to bring the working solution into the cell. This flow is visible due to the phenol red added to the working solution.
  3. We will also demonstrate direct injection into the cell cytoplasm. Direct microinjection into the cell is more robust but time-consuming as proper embryo orientation and microinjection technique is required. As the cell membrane is more rigorous than the yolk membrane, it is often quicker to enter the micropipette through the yolk to reach the cell cytoplasm. Orienting the animal pole towards the bottom of the trough and working with steady hands may aid in this method of injection.
  4. After microinjection, place the embryos into a Petri dish with E3 medium and incubate them at 28.5°C for normal development.
  5. Over the next several hours and days of embryo development, observe the embryos for your phenotypes of interest.

Part 8: Representative results

EGFP mRNA overexpression: To verify the success of the microinjection, we will monitor the expression of GFP in the developing embryos beginning at shield stage (~6 hpf) by in vivo whole-embryo fluorescence microscopy (Leica Microsystems GmBh, MZFLIII). Expression of the construct is most strong during the early events of embryonic development and will often diminish during development as the injected capped RNA is gradually degraded and depends on the stability of the expressed protein.

pkd2 translational-blocking morpholino: Based on previously published results, we expect the pkd2 morphants to mimic the dorsal body axis curvature as found in pkd2 mutant zebrafish and present kidney cysts at approximately 2 - 3 dpf4.

Figure 1
Figure 1: Representative results from microinjection of EGFP mRNA and pkd2 AUG morpholino. (a) TAB embryos were microinjected at the 1-cell stage with 0.17 ng of EGFP mRNA and visualized at 1 dpf for in vivo GFP expression. (b,c) TAB embryos were microinjected at the 1-cell stage with ~1.7 nl of a pkd2 translational blocking morpholino at 0.50 mM and scored for dorsal body curvature at 4 dpf.

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Microinjection into zebrafish embryos is a well-established and robust technique for exploring the role of a particular gene in development. Applications include overexpression, misexpression, and knockdown assays of your gene of interest as well as epistatic analysis between multiple genes. Microinjection in zebrafish has been widely used for generating transgenic animals, and mapping cell fate in early blastula embryos5,6,7,8,9. In addition, the application of this technique serves as a key step in generating germ-line chimeras by cell transplantation methods10.

One alternative method for exploring a gene’s ‘gain-of-function’ phenotype is to microinject constitutively active forms of that gene. In addition, a gene’s pseudo ‘loss-of-function’ phenotype may be explored by microinjection of dominant negative forms of that gene. Microinjection into zebrafish embryos may also include DNA and small molecules11,12.

A critical step in this microinjection technique is the quality of the micropipette needle. We make our micropipette from capillary tubes shaped by a pipette puller device. It is essential that the pipette puller is properly calibrated to yield optimal needle shape and size. Micropipettes that are too long and narrow often lack rigidity, break easily and struggle to penetrate the chorion and yolk. Short micropipettes are often more prone to damaging the embryo during microinjection.

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This work was supported by the NIH and PKD foundation to ZS. All animal experiments were conducted according to Yale Animal Resources Center (YARC) and Institutional Animal Care and Use Committee (IACUC) guidelines.


Name Type Company Catalog Number Comments
1x E3 Medium Reagent 5 mM NaCl
0.17 mM KCl
0.33 mM CaCl2
0.33 mM MgSO2
0.1% Methylene blue
All other materials are listed in the protocol.



  1. Westerfield, M. The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). 4th, University of Oregon. (2000).
  2. Nasevicius, A., Ekker, S. Effective targeted gene 'knockdown' in zebrafish. Nat Genet. 26, 216-220 (2000).
  3. Draper, B., Morcos, P., Kimmel, C. Inhibition of zebrafish fgf8 pre-mRNA splicing with morpholino oligos: a quantifiable method for gene knockdown. Genesis. 30, 154-156 (2001).
  4. Sun, Z., et al. A genetic screen in zebrafish identifies cilia genes as a principal cause of cystic kidney. Development. 131, 4085-4093 (2004).
  5. Stuart, G., McMurray, J., Westerfield, M. Replication, integration and stable germ-line transmission of foreign sequences injected into early zebrafish embryos. Development. 103, 403-412 (1988).
  6. Stuart, G., Vielkind, J., McMurray, J., Westerfield, M. Stable lines of transgenic zebrafish exhibit reproducible patterns of transgene expression. Development. 109, 577-584 (1990).
  7. Culp, P., Nüsslein-Volhard, C., Hopkins, N. High-frequency germ-line transmission of plasmid DNA sequences injected into fertilized zebrafish eggs. Proc Natl Acad Sci U S A. 88, 7953-7957 (1991).
  8. Strehlow, D., Heinrich, G., Gilbert, W. The fates of the blastomeres of the 16-cell zebrafish embryo. Development. 120, 1791-1798 (1994).
  9. Helde, K., Wilson, E., Cretekos, C., Grunwald, D. Contribution of early cells to the fate map of the zebrafish gastrula. Science. 265, 517-520 (1994).
  10. Lin, S., Long, W., Chen, J., Hopkins, N. Production of germ-line chimeras in zebrafish by cell transplants from genetically pigmented to albino embryos. Proc Natl Acad Sci U S A. 89, 4519-4523 (1992).
  11. Long, Q., et al. GATA-1 expression pattern can be recapitulated in living transgenic zebrafish using GFP reporter gene. Development. 124, 4105-4111 (1997).
  12. Murphey, R., Zon, L. Small molecule screening in the zebrafish. Methods. 39, 255-261 (2006).



  1. Dear
    Shiaulou Yuan & Zhaoxia Sun
    First of all thanks for such a nice presentation for the publication entitled "Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos". I am highly impressed by your work and the ease of understanding. I will be highly obliged if you could send me the full procedure of micro injection or alternatively full text of your publication. I am a PhD student working at Centre for Clinical Research in Neuropsychiatry,Graylands Hospital & Centre of Excellence for Alzhiemer's Disease Research & Care, Perth, Australia. I found this journal very useful for me. Do your university provide a short term training for some techniques related to Zebrafish Research. I shall be thankful to you for your kind help

    Posted by: Anonymous
    September 22, 2009 - 7:45 PM
  2. Hi Avdesh,
    Thank you for watching the video and for your comments. I can email you the PDF version of this protocol if you send me a email. Unfortunately, Yale dŒsn't seem to have any short-term zebrafish training courses. However, MBL Woods Hole usually offers a ²-week summer course titled "Zebrafish Development & Genetics." It seems like a nice opportunity to learn about zebrafish research with some great investigators & lecturers. Here's a link to their website if you're interested:
    Let me know if you have any other questions!

    Posted by: Anonymous
    September 23, 2009 - 3:37 PM
  3. Dear Shiaulou,
    Thanks for sharing the video, it helps lots for beginner like me. I encountered problem for my microinjection. I found accumulation of red liquid (which i suspect is phenol red) in the intestine of zebrafish embryos after 3 days of injection. I repeated the injection with different batch of embryos, i got the same result. So, i tried injecting morpholino without phenol red, i did not get any red liquid accumulation, this quite convince me that it was the phenol red in the intestine. So, do you come across this problem before? What usually happen to the phenol red after injection?

    Sze Huey

    Posted by: Tan S.
    May 11, 2010 - 11:40 PM
  4. Hi Sze,
    Thanks for watching! We have not encountered this problem before. Could it be due to a high injection amount of phenol red? Are your embryos visibly red at all several hours post injection? May I ask what dilution you're using and how much you're injecting? Also, we always obtain phenol red as a stock solution from Sigma - maybe a different source could the issue?

    From my understanding, phenol red has been widely used for years by numerous zebrafish labs (including ours) for aiding microinjections; it should be fairly innocuous. I always believed that the dye becomes diluted or pumped out of the cells gradually over time but I have not read any literature that examines that.

    One easy solution would be to inject without using phenol red since it's not necessary and only serves as a marker. Another idea is to use another dye, maybe bromophenol blue? I've never personally tried that but it could be an option!


    Posted by: Anonymous
    May 12, 2010 - 3:52 PM
  5. Thanks, Shiaulou.
    The phenol red I used was 0.²%, from stock solution of 0.5%, Sigma. I injected ².3nl (moprholino plus phenol red) into each embryo. Normally, the embryos are visibly red after ²-3 hours post injection.
    At which stage you think the elimination of the dye could happen? Because the accumulation of phenol red in the intestine can only be seen around 60-7² hours post fertilization.

    Sze Huey

    Posted by: Tan S.
    May 12, 2010 - 11:41 PM
  6. Hi Sze,
    The Sigma phenol red stock we obtain is also 0.5% but we use a final working concentration of 0.0²5% to 0.050% for injections. We take the the 0.5% Sigma stock and dilute it 1/10 or 1/²0 in our morpholino or RNA solution that we wish to inject. Is 0.²% phenol red your working concentration that you're injecting at? If so, that's could be the problem.

    I would guess that the phenol red injected at the 1-cell stage is gradually diluted as the cells divide and multiply but again, I have no evidence for this. Typically, I never notice the being visibly red ~7-8 hours post injection unless a huge amount or concentration of phenol red solution is injected. Is the dye gone from the intestine after day 3?


    Posted by: Anonymous
    May 15, 2010 - 3:22 PM
  7. Hi Shiaulou,
    Thanks for the reply. Yes, i injected 0.²% of the phenol red of the working concentration. The dye stayed in the intestine until day 7.

    Sze Huey

    Posted by: Anonymous
    May 19, 2010 - 1:57 AM
  8. Hi,
    First of all thanks for the video. In terms of results, what is the difference between injection on the yolk or directly into cell? Thanks in advance

    Posted by: Anonymous
    July 2, 2010 - 10:34 AM
  9. Hi,
    It's preferable to inject straight into the cell if you want to ensure maximum expression and phenotype penetrance. However, it's a little bit more convenient to inject into the yolk since you don't have to position the embryo as much. This can be advantageous if your experiment requires a lot of injected embryos (one example is for collecting lysate to do protein-work).

    Posted by: Anonymous
    July 2, 2010 - 4:17 PM
  10. Excellent. We also do routiinely in teh lab. Have you ever tried kupffer vesicle mounting and staining..... can you explain. I have tried several times (40-50 times) I succeeded only once!!

    Posted by: Suresh P.
    September 23, 2010 - 4:50 PM
  11. Hi,
    Yes, we do routine KV work. For staining and fixing KV for immunofluorescence, staging is quite is important; we recommend counting somites to ensure exact staging at 6-8 somite. Here's a protocol that has worked well for me:
    1. Dechorionate manually or by pronase treatment
    ². Stage the embryos for the age you desire (we recommend 6-8 somite)
    3. Fix O/N at 4 degrees using diluted formalin (1 part formalin to ².7 parts PBT)
    4. Wash off formalin with PBT x 3
    5. Manually deyolk using sharp watchmaker's forceps (#5) or tungsten needles. Be prepared to take your time! You want to remove all yolk from the embryo, this will improve your images greatly and makes the KV much easier to find.
    6. (Optional) Dehydrate into 100% MeOH (use 50/50 MeOH/PBT for 5' then 100% MeOH for 5') and incubate O/N at -²0 degrees. (Can skip this entire step if you desire, just depends on whether if your antibody requires MeOH fixation).
    7. Rehydrate your sample (50/50 MeOH/PBT for 5' then 100% PBT for 5').
    8. Wash 3x in PBT 5'
    9. (Optional) If you skipped MeOH step, treat the samples in pre-chilled acetone for 7' at -²0 degrees. Wash in 3x PBT again.
    9. Block for 30' to 1hr using blocking solution (10% FBS in PBT).
    10. Incubate O/N at 4 degrees or ²hrs at RT using your diluted primary antibody in blocking solution (for example, anti-acetylated tubulin + anti-atypical PKC are nice markers for ciliated KV work).
    11. Wash 5x PBT, ²0' each.
    1². Incubate O/N at 4 degrees or ²hrs at RT using your diluted secondary antibody in blocking solution.
    13. Wash 5x PBT, ²0' each.
    14. (Optional) Counterstain with DAPI, TOTO3 or equivalent (for example, 1:²0000 DAPI for ²0' in PBT at RT) then do a quick wash in PBT.
    15. Remove as much PBT as possible then add mounting medium and let equilibrate for ²0' at RT.
    16. Pipette onto a microscope slide and flat mount. Positioning is crucial, you want to lay the embryo perfectly flat with the ventral side facing you. Use nail polish to seal if desired.

    Feel free to contact me if you have more questions!


    Posted by: Anonymous
    September 23, 2010 - 6:15 PM
  12. Thanks for the reply. By the way, is there a "time" not stage or somites that helps in fixing. such as 1²hpf 14hpf etc....

    Posted by: Anonymous
    September 24, 2010 - 9:40 AM
  13. Hi,
    Time-wise, 6 somite is at 1²hpf, 8 somite is at 13hpf and 10 somite is 14hpf. Depending on your experiment, there could be developmental delay (some morpholinos and compounds show this effect) so I strongly recommend counting somites rather than solely relying on time if stage-matching your control and experimental embryos are important.


    Posted by: Anonymous
    September 25, 2010 - 8:38 PM
  14. OK

    Posted by: Guillermo L.
    November 18, 2010 - 2:06 PM
  15. Hi,

    Thanks for this video! Other protocols suggest injecting DNA/RNA/morpholino dissolved in KCl or Danieau's. Do you just use miliQ water, and do you think diluting in other solutions improves survival?


    Posted by: Anonymous
    January 24, 2012 - 10:18 AM
  16. Hi, I've tried Danieau's before and it dŒsn't seem to make a noticeable difference compared to water, except sometimes I've noticed that MO's in Danieau's can get cloudy or precipitate when in cold storage for a long time. Genetools themselves recommend dissolving in water but they also mention Ringer's or Danieau's as alternatives. From my observation, there dŒsn't seem to be a significant difference in survival between water or Danieau but if you're curious, you could always inject some wild type embryos with water and some others with Danieau to see. Thanks for watching

    Posted by: Anonymous
    January 24, 2012 - 2:27 PM
  17. Hi,i want to ask that what's your methods to get or extract proteins from 2 dpf /3dpf zebrafish embryos for western blot? does it need to remove chorionic when extract proteins from 3dpf embryos? i think that removing chorionic may make some proteins losted.

    Posted by: ma l.
    January 7, 2014 - 8:54 AM
  18. Hi, we basically use the protocol that the Heisenberg lab utilized in this paper:


    We follow their de-yolking protocol using pipette tips and then load the protein lysate onto gels using standard Western protocols. Use larger tips for older embryos. We do remove chorion. The Heisenberg protocol recommends doing so with pronase but I use watchmakers forceps. I haven't experienced any significant protein loss.


    Posted by: Anonymous
    January 8, 2014 - 9:18 AM
  19. Thanks very much for your reply! I want to consult another question: is the method to remove the yolk of embryos also follow that paper ? do you think this step is necessary? when I do western blot using proteins extracted from 3 dpf embryos, i didn't remove the yolk. I found removing the yolk would lost many proteins in the fish body.
    what's your lysis buffer? and how much volume add for per embryo?
    sincerely ,
    Lili Ma

    Posted by: ma l.
    January 8, 2014 - 9:54 AM

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