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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+as+a+model+for+complex+tissue+regeneration.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposon-mediated+BAC+transgenesis+in+zebrafish.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+perspectives+in+zebrafish+reverse+genetics:+moving+forward.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TALEN-mediated+precise+genome+modification+by+homologous+recombination+in+zebrafish.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+genome+editing+using+a+high-efficiency+TALEN+system.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromanipulation+of+gene+expression+in+the+adult+zebrafish+brain+using+cerebroventricular+microinjection+of+morpholino+oligonucleotides.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+electroporation+of+morpholinos+into+the+regenerating+adult+zebrafish+tail+fin.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+electroporation+of+morpholinos+into+the+adult+zebrafish+retina.">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., K&#246;ster, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantum+dots+are+powerful+multipurose+vital+labeling+agents+in+zebrafish+embryos.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+Pax-6+in+lens+regeneration.">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://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Zebrafish: a practical approach. 261, 121-143 (2000).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+morpholino+experiments:+don't+stop+making+antisense.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+primer+for+morpholino+use+in+zebrafish.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vectors+and+techniques+for+ectopic+gene+expression+in+zebrafish.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporally+controlled+site-specific+recombination+in+zebrafish.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fgf+signaling+instructs+position-dependent+growth+rate+during+zebrafish+fin+regeneration.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+models+for+cancer.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+Wnt+signaling+pathways+have+opposing+roles+in+appendage+regeneration.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+targets+of+the+Sox+domain+proteins+Dichaete+in+the+Drosophila+CNS+via+targeted+expression+of+dominant+negative+proteins.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Legless,+a+novel+mutation+found+in+PHT1-1+transgenic+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+DNA+rearrangements+and+early+developmental+arrest+caused+by+DNA+insertion+in+transgenic+mouse+embryos.">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 border="0" cellpadding="0" cellspacing="0" style="width:672px;" width="673">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:171px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Reagent/Material</td>
			<td style="width:171px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Morpholino with 3&#180;fluorescein tag</td>
			<td style="width:171px;">Gene Tools Inc.&#160;</td>
			<td style="width:119px;">Customized</td>
			<td style="width:167px;">More information on www.genetools.com</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Endo-Porter</td>
			<td style="width:171px;">Gene Tools Inc.&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">More information on www.genetools.com</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Agarose</td>
			<td style="width:171px;">Serva</td>
			<td style="width:119px;">120623</td>
			<td style="width:167px;">For pouring plates to place the fish during injections and imaging</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tricane (L-Ethyl-m-amino-benzate-methane sulfonate)</td>
			<td style="width:171px;">Applichem</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">For anesthesia</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">E3 medium (5 mM NaCl; 0.17 mM KCl; 0.33 mM CaCl2; 0.33 mM MgSo4)</td>
			<td style="width:171px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">For larval husbandry</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Petri Dishes</td>
			<td style="width:171px;">Sarstedt</td>
			<td style="width:119px;">821.472</td>
			<td style="width:167px;">For placing the fish during injections and imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#160;Equipment</td>
			<td style="width:171px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Thin Wall Glass Capillaries</td>
			<td style="width:171px;">World Precision Instruments, Inc.&#160;</td>
			<td style="width:119px;">TW100F-3</td>
			<td style="width:167px;">For preparing injection needles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microloader pipette tips</td>
			<td style="width:171px;">Eppendorf</td>
			<td style="width:119px;">5,242,956,003</td>
			<td style="width:167px;">For loading the needle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pasteur Pipettes (3 ml/1 ml)</td>
			<td style="width:171px;">Brand</td>
			<td style="width:119px;">747760/ 74775</td>
			<td style="width:167px;">For transfering larva</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Flaming / Brown&#160; needle puller&#160;</td>
			<td style="width:171px;">Sutter</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">More information on www.sutter.com</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Watchmaker (Dumont) Tweezers (size 5)</td>
			<td style="width:171px;">World Precision Instruments, Inc.&#160;</td>
			<td style="width:119px;">501985</td>
			<td style="width:167px;">To brake the needle before sharpening</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dissecting microscope</td>
			<td style="width:171px;">Olympus, Leica, Zeiss</td>
			<td style="width:119px;">Varies with the manufacturer</td>
			<td style="width:167px;">Part of the whole injection setup</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Fluorescence camera</td>
			<td style="width:171px;">Olympus, Leica, Zeiss</td>
			<td style="width:119px;">Varies with the manufacturer</td>
			<td style="width:167px;">To image the fluorescence after injeciton</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PicoNozzle kit (microinjector holder)</td>
			<td style="width:171px;">World Precision Instruments</td>
			<td style="width:119px;">5430-12</td>
			<td style="width:167px;">For microinjections</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pneumatic PicoPump (microinjector)</td>
			<td style="width:171px;">World Precision Instruments</td>
			<td style="width:119px;">SYS-PV820</td>
			<td style="width:167px;">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

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...

Watch this Scientific Journal Video about Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva at JoVE.com

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

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 ...

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

Research

JoVE Journal

Biology

10.5K Aufrufe.  Technische Universität Dresden.  Ein anpassungsfähiges reversen genetischen Methode für Zebrafisch-Gen-Funktion während der späteren Phasen der Entwicklung und physiologischen Homöostase wie Geweberegeneration unter Verwendung intraventrikuläre Injektion von Gen-spezifischen morpholinos beurteilen. 

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

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 ...

Zebrafish Brain Ventricle Injection

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved cavities within the brain that are filled with cerebrospinal fluid.  In zebrafish, after neural tube formation, the neuroepithelium undergoes a series of constrictions and folds while it fills with fluid resulting in brain ventricle formation.  In order to understand the process of ventricle formation, and the neuroepithelial shape changes that occur at the same time, we needed a way to visualize the ventricle space in comparison to the brain tissue.  However, the nature of transparent zebrafish embryos makes it difficult to differentiate the tissue from the ventricle space.  Therefore, we developed a brain ventricle injection technique where the ventricle space is filled with a fluorescent dye and imaged by brightfield and fluorescent microscopy.  The brightfield and the fluorescent images are then processed and superimposed in Photoshop.  This technique allows for visualization of the ventricle space with the fluorescent dye, in comparison to the shape of the neuroepithelium in the brightfield image.  Brain ventricle injection in zebrafish can be employed from 18 hours post fertilization through early larval stages.  We have used this technique extensively in our studies of brain ventricle formation and morphogenesis as well as in characterizing brain morphogenesis mutants (1-3).

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.

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved ...

Retro-orbital Injection in Adult Zebrafish

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is often the most effective and noninvasive form of delivery of an agent of interest. In the zebrafish (Danio rerio), IV injection of drugs has long been a challenge because of the small vessel diameter. This has also proved a significant hurdle for the injection of cells during hematopoeitic stem cell transplantation. Historically, injections into the bloodstream were done directly through the heart. However, this intra-cardiac procedure has a very high mortality rate as the heart is often punctured during injection leaving the fish prone to infection, massive blood loss or fatal organ damage. Drawing on our experience with the mouse, we have developed a new injection procedure in the zebrafish in which the injection site is behind the eye and into the retro-orbital venous sinus. This retro-orbital (RO) injection technique has been successfully employed in both the injection of drugs in the adult fish as well as transplantation of whole kidney marrow cells. RO injection has a much lower mortality rate than traditional intra-cardiac injection. Fish that are injected retro-orbitally tend to bleed less following injection and are at a much lower risk of injury to a major organ like the heart. Further, when performed properly, injected cells and/or drugs quickly enter the bloodstream allowing compounds to exert their effect on the whole fish and kidney cells to easily home to their niche. Thus, this new injection technique minimizes mortality while allowing efficient delivery of material into the bloodstream of adult fish. Here we exemplify this technique by retro-orbital injection of Tg(globin:GFP) cells into adult casper fish as well as injection of a red fluorescent dye (dextran, Texas Red ) into adult casper fish. We then visualize successful injections by whole animal fluorescence microscopy.

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

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage.
GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreERT). CreERT contains a modified estrogen receptor ligand binding domain which renders CreERT sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreERT, which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP.
Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreERT release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreERT.
We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders.
This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreERT;mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing ...

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In the zebrafish model, effective reverse genetic techniques have been developed using microinjection of gene-specific antisense morpholinos. Morpholinos target an mRNA through specific base-pairing and block gene function transiently by inhibiting translation or splicing for several days during embryogenesis (knockdown). However, in vertebrates such as mouse or zebrafish, some gene functions can be obscured by these approaches due to the presence of another gene that compensates for the loss. This is especially true for gene families containing sister genes that are co-expressed in the same developing tissues. In zebrafish, functional compensation can be tested in a relatively high-throughput manner, by co-injection of morpholinos that target knockdown of both genes simultaneously. Likewise, using morpholinos, a genetic interaction between any two genes can be demonstrated by knockdown of both genes together at sub-threshold levels. For example, morpholinos can be titrated such that neither individual knockdown generates a phenotype. If, under these conditions, co-injection of both morpholinos causes a phenotype, a genetic interaction is shown. Here we demonstrate how to show functional redundancy in the context of two related GATA transcription factors. GATA factors are essential for specification of cardiac progenitors, but this is revealed only by the loss of both Gata5 and Gata6. We show how to carry out microinjection experiments, validate the morpholinos, and evaluate the compensated phenotype for cardiogenesis.

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.

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A Multi-hole Cryovial Eliminates Freezing Artifacts when Muscle Tissues are Directly Immersed in Liquid Nitrogen

Studies on skeletal muscle physiology face the technical challenge of appropriately processing the specimens to obtain sections with clearly visible cytoplasmic compartments. Another hurdle is the tight apposition of myofibers to the surrounding tissues. Because the process of tissue fixation and paraffin embedding leads to the shrinkage of muscle fibers, freezing is an optimal means of hardening muscle tissue for sectioning. However, a commonly encountered issue, the formation of ice crystals, occurs during the preparation of frozen sections because of the high water content of muscle. The protocol presented here first describes a simple and efficient method for properly freezing muscle tissues by immersing them in liquid nitrogen. The problem with using liquid nitrogen alone is that it causes the formation of a nitrogen gas barrier next to the tissue, which acts as an insulator and inhibits the cooling of the tissues. To avoid this "vapor blanket" effect, a new cryovial was designed to increase the speed of liquid flow around the tissue surface. This was achieved by punching a total of 14 inlet holes in the wall of the vial. According to bubble dynamics, a higher rate of liquid flow results in smaller bubbles and fewer chances to form a gas barrier. When liquid nitrogen flows into the cryovial through the inlet holes, the flow velocity around the tissue is fast enough to eliminate the gas barrier. Compared to the method of freezing muscle tissues using pre-chilled isopentane, this protocol is simpler and more efficient and can be used to freeze muscle in a throughput manner. Furthermore, this method is optimal for institutions that do not have access to isopentane, which is extremely flammable at room temperature.

This protocol describes a procedure to freeze muscle tissues by plunging them directly into liquid nitrogen. This protocol also highlights a new cryovial that can avoid the &#34;blanket effect&#34; of nitrogen gas when liquid nitrogen contacts the tissue surface of a specimen.

Studies on skeletal muscle physiology face the technical challenge of appropriately processing the specimens to obtain sections with clearly visible ...