Name: Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva (Video) | JoVE Journal
Uploaded: 2014-06-17T15:00:00.000+00:00
Duration: 8 min 22 s
Description: Learn a reverse genetic morpholino method for studying gene function during zebrafish fin regeneration using cardiac ventricular injection. This technique enables knockdown in difficult-to-target larval tissues.

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5 minutes

This work was supported by the Deutsche Forschungsgemeinschaft (DFG), we wish to thank Ayele Tsedeke Taddese for technical support.

1. Preparation of the Glass Needles (Figure 1)
<ol>
	<li>Use glass capillaries with a 0.75 mm diameter to prepare injection needles (Figure 1A).</li>
	<li>Place the glass capillary into a needle puller and pull the needle with the following parameter: heating cycle value: 463; pulling cycle value: 230; velocity: 150 msec; time: 150 msec (Figure 1B).</li>
	<li>Break the pulled glass capillary with watchmaker tweezers to produce a 20 &#181;m diameter needle under a stereoscope with a micrometer eyepiece.</li>
	<li>Use a lathe with a wetted rubber spinning wheel to sharpen the needle and produce a 20 &#181;m bevel (Figures 1C and 1D). Note: Sharp, beveled needles improve the ease of insertion into the ventricle and thus minimize the damage to the tissue and allow the perforated muscle to reseal after removal of the needle (Figures 1E-G).</li>
</ol>
2. Preparation of the Morpholino Solution
<ol>
	<li>Prepare the morpholino stock by dissolving the lyophilized morpholino in 1x Phosphate-Buffered Saline (PBS) to a final concentration of 7.5 mM. [See manufacturer&#39;s instructions for details (Materials table)].</li>
	<li>Prepare the morpholino injection solution by mixing 2.5 &#181;l morpholino stock solution (7.5 mM) with 2.8 &#181;l Endo-Porter stock solution (1 mM) (See&#160;Materials table) for a final concentration of 3.5 mM morpholino and 0.5 mM Endo-Porter.</li>
</ol>
3. Preparation of the Morpholino Injection (Figure 2)
<ol>
	<li>Load the beveled glass needle with 5 &#181;l of this solution using a micropipette with a 10 &#181;l microloader pipette tip.</li>
	<li>Insert the glass needle into the needle holder of the micromanipulator connected to the pneumatic pico pump (Figures 2A-C).</li>
	<li>Place the needle holder next to the microscope so that the needle only needs to be moved in one planar direction to insert it into the cardiac ventricle of the larva (Figure 2A).</li>
	<li>Adjust the angle for the injections at around 45&#176;.</li>
	<li>Set the microinjector values as follows: hold pressure: 20 pounds per square inch (psi); ejection pressure: 15 psi; 100 msec range of gating, period value of 1.9 (corresponds to 10.9 msec).</li>
	<li>Melt agarose in 1x PBS using a standard microwave to produce 20 ml of a 1.5% (w/v) gel. Pour melted gel into a 10 cm Petri dish. Place a grooved injection form into the warm agarose so that once hardened, the agarose will have furrows in which the sedated larva will be placed11 (Figures 2D and 2E).</li>
</ol>
4. Injections (Figure 3)
<ol>
	<li>Anesthetize larva in 100 ml of aquarium water with 20 mg/L tricaine (L-Ethyl-m-amino-benzoate-methane sulfonate) until they stop responding to touch.</li>
	<li>Use a plastic Pasteur pipette to carefully transfer the sedated larva into a groove of the wet agarose mold so that the ventral side is facing against the vertical agarose wall of the groove (Figure 3A).</li>
	<li>Place the agarose plate under the stereomicroscope so that the ventricle is facing away from the injection needle (Figure 3A).</li>
	<li>Lower the needle to insert it into the heart ventricle. Insert the needle only 1-2 &#181;m into the ventricle taking care not to insert the needle too deeply (Figures 3B and 3C).</li>
	<li>Once the needle is inserted, inject the morpholino solution into the ventricle with 4-6 pulses, each delivering 3 nl of the solution, with waiting intervals to allow clearing of the heart (Figures 3D and 3E).</li>
	<li>After injection, remove the needle (parallel to the plane of insertion) and then carefully transfer the larva using a plastic Pasteur pipette filled with E3 medium back into a Petri dish containing fresh E3 medium.</li>
	<li>Place the needle into a Petri dish containing 1x PBS to prevent the drying of the needle when transferring the injected larva.</li>
	<li>Repeat steps 4.1-4.6 every 12-24 hr&#160;for the duration of the experiment. Note: Repeating the injections ensures the uptake and maintenance of the morpholino in cells.</li>
</ol>
5. Analyses of Injections (Figure 4)
<ol>
	<li>Anesthetize the fish in 100 ml of aquarium water with 20 mg/L tricaine until they stop responding to touch.</li>
	<li>Place the larva laterally on a flat wet 1.5% (w/v) agarose plate covered with E3 medium.</li>
	<li>Image larvae using a stereomicroscope with brightfield and fluorescence imaging. Note: As the larvae are transparent, the fluorescein-tagged morpholino should be visible as fluorescent green in the blood vessels throughout the animal directly after injection. This fluorescence will further disperse into the vascularized tissues by 15 min&#160;(Figures 4A-4C).</li>
</ol>
6. Assessment of Regenerative Outgrowth
<ol>
	<li>Assess captured images using Fiji Image J free software (http://fiji.sc/Fiji). For investigating regeneration, use the line-tracing tool to determine the amount of regenerative growth that has occurred on control and morpholino-injected larva.</li>
	<li>To calibrate images, open one of the original image files in Fiji by dragging and dropping the file into the main Fiji window.</li>
	<li>To set the scale for all following images, go to &#34;Analyze&#34; in the main menu.</li>
	<li>In the drop down menu click on &#34;Set scale&#34;.</li>
	<li>In this window, turn on the &#34;Global&#34; option by clicking the check box.</li>
	<li>Now open the image to measure the amount of regenerative growth again by drag and drop (see step 6.1).</li>
	<li>Choose the &#34;freehand selections&#34; tool in the tool bar.</li>
	<li>Encircle the area to be measured (Figures&#160;4J-4L).</li>
	<li>Press Ctrl+M. This will open a new &#34;Results&#34; window showing the value of the encircled area in square pixels.</li>
</ol>

<ol><li>Gemberlin, M., Bailey, T. J., Hyde, D. R., Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+zebrafish+as+a+model+for+complex+tissue+regeneration%5BTitle%5D)+AND+%22Trends+in+Genetics%22%5BJournal%5D)">The zebrafish as a model for complex tissue regeneration.</a> Trends in Genetics. 29, 611-620 (2013).</li>
<li>Suster, M. L., Abe, G., Schouw, A., Kawakami, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transposon-mediated+BAC+transgenesis+in+zebrafish%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Transposon-mediated BAC transgenesis in zebrafish.</a> Nature Protocols. 6, 2011 (1998).</li>
<li>Skromne, I., Prince, V. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Current+perspectives+in+zebrafish+reverse+genetics%3A+moving+forward%5BTitle%5D)+AND+%22Developmental+Dynamics%22%5BJournal%5D)">Current perspectives in zebrafish reverse genetics: moving forward.</a> Developmental Dynamics. 237, 861-882 (2008).</li>
<li>Zu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TALEN-mediated+precise+genome+modification+by+homologous+recombination+in+zebrafish%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">TALEN-mediated precise genome modification by homologous recombination in zebrafish.</a> Nature Protocols. 10, 329-332 (2013).</li>
<li>Bedell, V. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+genome+editing+using+a+high-efficiency+TALEN+system%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">In vivo genome editing using a high-efficiency TALEN system.</a> Nature. 491, 114-118 (2012).</li>
<li>Kizil, C., IItzsche, A., Kaslin, J., Brand, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Micromanipulation+of+gene+expression+in+the+adult+zebrafish+brain+using+cerebroventricular+microinjection+of+morpholino+oligonucleotides%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Micromanipulation of gene expression in the adult zebrafish brain using cerebroventricular microinjection of morpholino oligonucleotides.</a> J Vis Exp. , (2013).</li>
<li>Hyde, D., Godwin, A., Thummel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+electroporation+of+morpholinos+into+the+regenerating+adult+zebrafish+tail+fin%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">In vivo electroporation of morpholinos into the regenerating adult zebrafish tail fin.</a> J Vis Exp. , (2012).</li>
<li>Thummel, R., Bailey, T., Hyde, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+electroporation+of+morpholinos+into+the+adult+zebrafish+retina%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">In vivo electroporation of morpholinos into the adult zebrafish retina.</a> J Vis Exp. , (2011).</li>
<li>Rieger, S., Kulkarni, R. P., Darcy, D., Fraser, S. E., Köster, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantum+dots+are+powerful+multipurose+vital+labeling+agents+in+zebrafish+embryos%5BTitle%5D)+AND+%22Developmental+Dynamics%22%5BJournal%5D)">Quantum dots are powerful multipurose vital labeling agents in zebrafish embryos.</a> Developmental Dynamics. 234, 670-681 (2005).</li>
<li>Madhavan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+Pax-6+in+lens+regeneration%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Science+U%22%5BJournal%5D)">The role of Pax-6 in lens regeneration.</a> Proceedings of the National Academy of Science U. 103, 14848-14853 (2006).</li>
<li>Gilmour, D. T., Jessen, J. R., Lin, S. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aGilmour%2C+DT+%22Zebrafish%3A+a+practical+approach%22">Zebrafish: a practical approach</a>. 261, University Press. Oxford. 121-143 (2000).</li>
<li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fiji%3A+an+open-source+platform+for+biological-image+analysis%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9, 676-682 (2012).</li>
<li>Eisen, J. S., Smith, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Controlling+morpholino+experiments%3A+don%27t+stop+making+antisense%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Controlling morpholino experiments: don't stop making antisense.</a> Development. 135, 1735-1743 (2008).</li>
<li>Bill, B. R., Petzold, A. M., Clark, K. J., Schimmenti, L. A., Ekker, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+primer+for+morpholino+use+in+zebrafish%5BTitle%5D)+AND+%22Zebrafish%22%5BJournal%5D)">A primer for morpholino use in zebrafish.</a> Zebrafish. 6, 69-77 (2009).</li>
<li>Hyatt, T. M., Ekker, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vectors+and+techniques+for+ectopic+gene+expression+in+zebrafish%5BTitle%5D)+AND+%22Methods+Cell+Biol%22%5BJournal%5D)">Vectors and techniques for ectopic gene expression in zebrafish.</a> Methods Cell Biol. 59, 117-126 (1999).</li>
<li>Hans, S., Kaslin, J., Freudenreich, D., Brand, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Temporally+controlled+site-specific+recombination+in+zebrafish%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Temporally controlled site-specific recombination in zebrafish.</a> PLoS One. 4, (2009).</li>
<li>Lee, Y., Grill, S., Sanchez, A., Murphy-Ryan, M., Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fgf+signaling+instructs+position-dependent+growth+rate+during+zebrafish+fin+regeneration%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Fgf signaling instructs position-dependent growth rate during zebrafish fin regeneration.</a> Development. 132, 5173-5183 (2005).</li>
<li>Liu, S., D, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zebrafish+models+for+cancer%5BTitle%5D)+AND+%22Annu.+Re.+Pathol.+Mech.+Dis%22%5BJournal%5D)">Zebrafish models for cancer.</a> Annu. Re. Pathol. Mech. Dis. 6, 71-93 (2011).</li>
<li>Stoick-Cooper, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distinct+Wnt+signaling+pathways+have+opposing+roles+in+appendage+regeneration%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Distinct Wnt signaling pathways have opposing roles in appendage regeneration.</a> Development. 134, 479-489 (2007).</li>
<li>Shen, S. P., Aleksic, J., Russell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identifying+targets+of+the+Sox+domain+proteins+Dichaete+in+the+Drosophila+CNS+via+targeted+expression+of+dominant+negative+proteins%5BTitle%5D)+AND+%22BMC+Dev.+Biol%22%5BJournal%5D)">Identifying targets of the Sox domain proteins Dichaete in the Drosophila CNS via targeted expression of dominant negative proteins.</a> BMC Dev. Biol. 13, (2013).</li>
<li>McNeish, J. D., Scott, W. J., Potter Jr, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Legless%2C+a+novel+mutation+found+in+PHT1-1+transgenic+mice%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Legless, a novel mutation found in PHT1-1 transgenic mice.</a> Science. 241, 837-839 (1988).</li>
<li>Covarrubias, L., Nishida, Y., Terao, M., D'Eustachio, P., Mintz, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellular+DNA+rearrangements+and+early+developmental+arrest+caused+by+DNA+insertion+in+transgenic+mouse+embryos%5BTitle%5D)+AND+%22Mol.+Cell.+Biol%22%5BJournal%5D)">Cellular DNA rearrangements and early developmental arrest caused by DNA insertion in transgenic mouse embryos.</a> Mol. Cell. Biol. 7, 2243-2247 (1987).</li>
</ol>

The authors have nothing to disclose.

The intraventricular injection provides a quick and reliable assessment method for testing gene function at later stages of development or of body homeostasis without affecting the gene function during embryogenesis. To ensure the success of this technique, one should be aware of four critical points: 1) needle size, 2) drying out of the needle, 3) minimizing volume, and 4) minimal exposure time in the sedation solution. Needles that are too small&#160;will clog frequently, while needles that are too large will damage th...

Regeneration of organs and appendages is fundamentally important for survival and fitness; however, several vertebrates including man have limited regenerative abilities. While several animal models exist that have extensive regenerative capacity, reverse genetic techniques to assess gene function during organ and appendage regeneration remain very limited or non-existent. Therefore, new approaches are required to dissect the molecular biology of regeneration in these model organisms.
The zebrafish is a well-established model for understanding the cell and molecular biology of organ and appendage regeneration1, not only because of its significant ability to regenerate multiple organs, tissues and appendages, but also because several transgenic fish lines exist to track cells and to overexpress gene constructs2,3. However, gene inhibition in larval zebrafish is mainly limited to the overexpression of dominant-negative constructs, which are not available to all genes of interest or whose transgene product can acquire gain-of-function effects that do not reflect the endogenous activity of the gene. Thus, an alternative method to specifically remove gene expression by knockout or knockdown is needed to overcome these issues.
Gene-specific targeting using TALENS exists as a reverse genetic means to knockout gene function; however, this knockout strategy is very frequently limited to functional assessments during early embryogenesis, because initial requirements of the gene prevent further progression of embryonic development. Thus, studying later phenomena such as regeneration or organ homeostasis after development using TALENS is precluded4,5. Therefore, an alternate gene removal strategy is needed that targets gene function after early development to assess gene requirements in fully formed organs and structures.
Morpholino injection has been shown to be effective in targeting genes in a few adult organs and the adult regenerating fin6-8, but these methods require electroporation and many internal organs are difficult to electroporate either due to their location or due to their sensitivity to electrical disruption. Furthermore, some tissues in the larva are difficult to inject directly, because direct injection may disrupt their structural integrity or because their size is limiting. The caudal fin of the larva is one such structure, because direct injection into the finfold is not possible. Thus, an alternative to electroporation and direct injection was needed to target genes in tissues that are either too small to inject or can&#39;t be electroporated.
In order to target and inhibit the function of specific genes during the regeneration of the larval caudal fin, we have modified existing morpholino technologies allowing the assessment of gene function during caudal fin regeneration in late-staged larva. This method employs intraventricular delivery9 of fluorescein-tagged morpholinos together with Endo-Porter transfection reagent10. Once in the ventricle, the morpholino-Endo-Porter mixture quickly spreads throughout the larva via the vasculature and enters tissues that have been previously impossible to target. This injection method may be modified to target genes in specific tissues and quite possibly can be applied in other animal models that currently lack reverse genetic methods to inhibit gene function. Thus, it offers a quick and easy method with the potential for broad range use to immediately study gene function during general organ homeostasis and regeneration at larval stages.

<table><tbody><tr><td>Morpholino with 3&amp;acute;fluorescein tag</td><td>Gene Tools Inc.</td><td>Customized</td><td>More information at www.gene-tools.com</td></tr><tr><td>Endo-Porter</td><td>Gene Tools Inc.</td><td></td><td>More information at www.gene-tools.com</td></tr><tr><td>Agarose</td><td>Serva</td><td>120623</td><td>For pouring plates to place the fish during injections and imaging</td></tr><tr><td>Tricaine (L-Ethyl-m-amino-benzate-methane sulfonate)</td><td>Applichem</td><td></td><td>For anesthesia</td></tr><tr><td>E3 medium (5 mM NaCl; 0.17 mM KCl; 0.33 mM CaCl&lt;sub&gt;2&lt;/sub&gt;; 0.33 mM MgSO&lt;sub&gt;4&lt;/sub&gt;)</td><td></td><td></td><td>For larval husbandry</td></tr><tr><td>Petri Dishes</td><td>Sarstedt</td><td>821.472</td><td>For placing the fish during injections and imaging</td></tr><tr><td>Thin Wall Glass Capillaries</td><td>World Precision Instruments, Inc.</td><td>TW100F-3</td><td>For preparing injection needles</td></tr><tr><td>Microloader pipette tips</td><td>Eppendorf</td><td>5,242,956,003</td><td>For loading the needle</td></tr><tr><td>Pasteur Pipettes (3 ml/1 ml)</td><td>Brand</td><td>747760/ 74775</td><td>For transfering larva</td></tr><tr><td>Flaming/Brown needle puller</td><td>Sutter</td><td></td><td>More information at www.sutter.com</td></tr><tr><td>Watchmaker (Dumont) Tweezers (size 5)</td><td>World Precision Instruments, Inc.</td><td>501985</td><td>To brake the needle before sharpening</td></tr><tr><td>Dissecting microscope</td><td>Olympus, Leica, Zeiss</td><td>Varies with the manufacturer</td><td>Part of the whole injection setup</td></tr><tr><td>Fluorescence camera</td><td>Olympus, Leica, Zeiss</td><td>Varies with the manufacturer</td><td>To image the fluorescence after injeciton</td></tr><tr><td>PicoNozzle kit (microinjector holder)</td><td>World Precision Instruments</td><td>5430-12</td><td>For microinjections</td></tr><tr><td>Pneumatic PicoPump (microinjector)</td><td>World Precision Instruments</td><td>SYS-PV820</td><td>For microinjections</td></tr></tbody></table>

reverse genetic morpholino approach using cardiac ventricular injection to transfect multiple difficult-to-target tissues in the zebrafish larva

Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva

The zebrafish is an important model to understand the cell and molecular biology of organ and appendage regeneration. However, molecular strategies to employ reverse genetics have not yet been adequately developed to assess gene function in regeneration or tissue homeostasis during larval stages after zebrafish embryogenesis, and several tissues within the zebrafish larva are difficult to target. Intraventricular injections of gene-specific morpholinos offer an alternative method for the current inability to genomically target zebrafish genes in a temporally controlled manner at these stages. This method allows for complete dispersion and subsequent incorporation of the morpholino into various tissues throughout the body, including structures that were formerly impossible to reach such as those in the larval caudal fin, a structure often used to noninvasively research tissue regeneration. Several genes activated during larval finfold regeneration are also present in regenerating adult vertebrate tissues, so the larva is a useful model to understand regeneration in adults. This morpholino dispersion method allows for the quick and easy identification of genes required for the regeneration of larval tissues as well as other physiological phenomena regulating tissue homeostasis after embryogenesis. Therefore, this delivery method provides a currently needed strategy for temporal control to the evaluation of gene function after embryogenesis.&#160;

The sharp, beveled injection needle is easily placed in the cardiac ventricle of the zebrafish larva when approached dorsally (Figure 3A). The heart continues pumping and blood flow is maintained despite the presence of the needle (Figures 3B and 3C). Careful injections do not disrupt the morphology of the ventricle or cardiac contractions (Figure 3D) despite injection of the morpholino into the heart (Figure 3E).
<p class="jove_cont...

An adaptable reverse genetic method for zebrafish to assess gene function during later stages of development and physiological homeostasis such as tissue regeneration using intraventricular injections of gene-specific morpholinos.

The zebrafish is an important model to understand the cell and molecular biology of organ and appendage regeneration. However, molecular strategies to ...

The overall goal of this procedure is to deliver morph eno oligonucleotides via ventricular injections into the larval, coddle fin, and knock down genes to assess their function during regeneration. This is accomplished by first anesthetizing the larva and placing them into a groove of the aros mold. Next, the injection needle is inserted into the heart ventricle.Then the morpho solution is injected four to six times into the ventricle with weighting intervals between pulses to allow clearing of the heart. Finally, the coddle fin is amputated and allowed to regenerate for three days. Ultimately, the effect on fin regeneration can then be assessed by measuring the regenerated area of the fin using the line tracing tool of Fiji image J open source software.The main advantage of this technique over existing methods like transgenesis or mutagenesis, is that it allows the knockdown of genes by minos in larval tissues that are not suitable for direct injections Using glass capillaries with a 0.75 millimeter diameter, place one into a needle puller and pull the needle with the following parameters with watchmakers tweezers, and under a stereoscope with a micrometer eyepiece, break the pulled capillary to produce a 20 micrometer diameter needle. Then use a lathe with a wedded rubber spinning wheel to sharpen the needle and produce a 20 micrometer bevel. Prepare the morpho stock by dissolving the Lyophilized Morpho in one XPBS to a final concentration of 7.5 millimolar.Mix the Morpho injection solution by combining 2.5 microliters of Morpho stock solution with 2.8 microliters of one millimolar endo Porter stock solution for a final concentration of 3.5 millimolar morph and 0.5 millimolar endo porter to carry out injections. Use a micro pipette with a 10 microliter tip to load the beveled glass needle with five microliters of morpho solution. Then insert the glass needle into the needle holder of the micro manipulator connected to the pneumatic pico pump.Adjust the angle for the injections to approximately 45 degrees so that the needle only needs to be moved in one planer direction. Next, set the micro injector values as follows, A hold pressure of 20 pounds per square inch or PSI an ejection pressure of 15 PSIA 100 millisecond range of gating and a period value of 1.9, which corresponds to 10.9 milliseconds. Prepare 20 milliliters of melted 1.5%aros in one XPBS and pour it into a 10 centimeter petri dish.Then place a grooved injection mold into the warm aros to form furrows in the hardened gel to anesthetize larvae. Place them into 100 milliliters of aquarium water with 20 milligrams per liter of trican. After they stop responding to touch, use a plastic pastoral pipette to carefully transfer the larva into a groove of the wet aros mold so that the ventral side is facing the vertical aros wall of the groove.Place the aros plate under the stereo microscope so that the ventricle is facing away from the injection needle. Next, lower the needle and insert it approximately one to two micrometers into the heart ventricle. Taking care not to insert it too deeply.Then inject a three nanoliter pulse of Morpho solution into the ventricle and weight to allow clearing of the heart before repeating with five more pulses after injection, remove the needle and place it into a Petri dish containing one XPBS to prevent drying. Then using a transfer pipette filled with E three medium carefully transfer the larvae back into a Petri dish containing fresh E three medium to ensure uptake and maintenance of the morpho in cells. Repeat the injections every 12 to 24 hours for the duration of the experiment to assess the injections.After anesthetizing the fish, place them on a flat wet aros plate covered with E three medium before using a stereo microscope with bright field and fluorescence to image them. Analyze images for regeneration using Fiji Image J free software to use the line tracing tool to determine the amount of regenerative growth that has occurred. First, calibrate the images by dragging and dropping a file into the main Fiji window in the main menu.Set the scale for all images by clicking on analyze in the dropdown menu. Click set scale, then turn on the global option by clicking the checkbox. Now drag and drop the image again to measure the amount of regenerative growth in the toolbar.Choose the freehand selections tool and encircle the area to be measured. Finally, press Ctrl M.This will open a new results window showing the value of the encircled area in square pixels. As seen here within minutes of injection, the morpho endo porter solution distributes throughout the vasculature into different tissues such as the fin fold and brain for at least 12 or more hours.To assess the regeneration of distal larval fin structures, the following were removed, the distal tip of the fin fold and spinal cord, the distal no cord, distal trunk muscle, which is not visible here, and pigment cells, which are difficult to target by direct injection, but appear to incorporate the morpho after serial ventricular injections. Thus, this method relatively easily promotes the delivery of morpho to tissues that are difficult to individually target, and it permits the assessment of gene function in several tissues in the regenerating fin at once. To analyze the importance of specific genes involved in regeneration, the larval coddle fin is partially amputated, and all the tissues that have incorporated the morpho are resected.The larval fin fold regenerates its structure within a few days post amputation as demonstrated. Here, morpho is targeting genes required for regeneration, perturbed the regeneration response compared to unin injected and mismatch morpho controls controls After its development. This technique paved the way for researchers in the field of regeneration to explore the molecular mechanisms that are involved in regeneration of the zebra fish Fin.

reverse-genetic-morpholino-approach-using-cardiac-ventricular

Research

JoVE Journal

Biology

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.

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

Gene function can be obscured in loss-of-function experiments if there is compensation by another gene. The zebrafish model provides a relatively high-throughput means to reveal such functional redundancy in living embryos.

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

A method to conditionally knockdown a target protein’s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein’s role in the regenerating or intact retina.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

The protocol describes targeted knockdown of gene expression in zebrafish by morpholino injection in the tail.

Morpholino Injection: A Method to Knockdown Target Gene Expression to Study Function in Zebrafish

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

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.

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

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

This work describes a protocol for the modular Tol2 transgenesis system, a gateway-based cloning method to create and inject transgenic constructs into zebrafish embryos.

The Zebrafish Tol2 System: A Modular and Flexible Gateway-Based Transgenesis Approach

After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles.  We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.

Zebrafish Brain Ventricle Injection

Here we show how to do retro-orbital injection in adult zebrafish.

Retro-orbital Injection in Adult Zebrafish

Learn a reverse genetic morpholino method for studying gene function during zebrafish fin regeneration using cardiac ventricular injection. This technique enables knockdown in difficult-to-target larval tissues.

Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva (Video) | JoVE Journal

Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva

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