The authors would like to thank the Office of Research of Presbyterian College School of Pharmacy for financial support of this project through the Small Pharmacy Awards for Research &#38; Collaboration (SPARC) Award. The authors thank Mr. John Smink, Manager of the Aquatic Animal Research Laboratory, Clemson University, Clemson, SC for his technical assistance manuscript revision.

NOTE: These protocols follow the guidelines of IACUC of Presbyterian College.
1. Acquire Materials Listed in Table 1.
2. Assemble the 5 Shelf Chrome According to the Manufacturer&#8217;s Instructions. This will be used to House the Facility.
3. Construct the water reservoir and filtration system (Figures 1A and 2).
<ol>
	<li>Using Figure 1A as a guide, layout the 18 gallon bucket, pump, filters and UV lamp to determine final installation location and size.</li>
	<li>Once parts are laid out, measure and cut the &#190;&#8221; PVC pipe to the length needed for assembly. Vary the lengths of all pipes according to specific space needs and limitations.</li>
	<li>Assemble the pipes and fittings as laid out previously using the PVC primer and cement, see below (3.3.2 and 3.3.3).
	<ol>
		<li>Sand the cut edges of the pipe with sandpaper until they are smooth to the touch and there is no debris.</li>
		<li>Prime the outside of the pipe and the inside of the fitting with the purchased PVC primer with the included swab from the PVC primer.</li>
		<li>Cement the pipe and fitting together by applying the PVC cement to the outside of the pipe using the included swab. Repeat for all steps that need PVC cementing.</li>
		<li>Place the 18 gallon bucket to one side of the shelf construction.</li>
		<li>Install a bulkhead connector in the side of the reservoir tank.</li>
		<li>Attach a &#190;&#8221; PVC pipe to the bulkhead.</li>
		<li>Attach the other end of the &#190;&#8221; pipe to the pump inlet using a &#190;&#8221; threaded make fitting.</li>
		<li>Connect the filters to the pump using two 90 degree elbows and &#190;&#8221; PVC pipe.
		<ol>
			<li>Use one paper filter followed by one activated carbon filters in order to filter out debris and reduce chlorine and chemical levels in the water.</li>
		</ol>
		</li>
		<li>Connect the UV lamp to the filters according to the manufacturer&#8217;s instructions.</li>
	</ol>
	</li>
	<li>Attach the pump and filter system to the top side of the first shelf of the rack using wire ties. Vary the order of the assembly and attachment according to the size and layout of the shelf/rack chosen.</li>
	<li>Add 1 bioball per gallon of water to the water reservoir once the pump and filter system are assembled and attached to the shelf.</li>
</ol>
4. Construct the main supply line (Figures 1B and 2).
<ol>
	<li>Measure and cut a piece of &#190;&#8221; PVC pipe to connect the UV lamp to the main supply</li>
	<li>Use a &#190;&#8221; PVC tee to start the main supply line by connecting the tee to the &#190;&#8221; PVC exhaust coming from the UV filter. .
	<ol>
		<li>Add an on-off valve to the bottom of the PVC tee to serve as a drain for maintenance flushing of the system.</li>
		<li>Start the main supply line from the top of the &#190;&#8221; PVC tee by measuring and cutting &#190;&#8221; PVC pipe to desired length to provide feed pipes for each of the shelves on the rack.</li>
		<li>Install &#190;&#8221; PVC tees for the horizontal lines that will enter each shelf of the rack.</li>
		<li>Optionally (and suggested), add a &#190;&#8221; on-off valve for each shelf supply line to allow for isolation of each shelf and a modular design of the system.</li>
	</ol>
	</li>
	<li>Use a &#190;&#8221; PVC pipe as a pressure reducing line on the top shelf. If this line runs on the back of the shelf, use this shelf for storage. End the pressure reducing line in the main exhaust line (Figure 1B) with a &#190;&#8221; on-off valve.</li>
	<li>Once the above steps have been completed, prime and cement the PVC pipe together as previously done when constructing the reservoir and filtration system.</li>
</ol>
5. Construct the main exhaust line (Figures 1C and 2).
<ol>
	<li>Run the vertical length of the main exhaust line to the other side of the rack from the main supply line. Use a 2&#8221; PVC pipe for this vertical line to receive the main supply line at the top and drain lines from the fish tanks from each shelf.</li>
	<li>Cut the pieces of 2&#8221; PVC pipe for the vertical main exhaust line and use 2&#8221; PVC tees to receive the fish tank drain lines from each shelf. End the main exhaust line in the stock tank to expel the recycled water over a sponge filter.</li>
	<li>Cut a one inch opening the entire length of the 2&#8221; PVC pipe pieces and attach to vertical main exhaust line with PVC tees for the horizontal drain lines from the fish tanks.</li>
	<li>Cap the 2&#8221; horizontal drain lines with a 2&#8221; PVC cap.</li>
	<li>Prime and cement the pieces using the necessary fittings (connectors, nipples or elbows).</li>
</ol>
6. Construct the fish tanks (Figure 3).
<ol>
	<li>With the drill, cut a 1 1/8&#8221; hole at the back of the fish tank 1&#8221; below the upper edge with a hole saw.</li>
	<li>Slide a threaded &#190;&#8221; PVC male adapter (with a #18 O ring attached) through the hole (from the inside of the tank to the outside).</li>
	<li>Attach and tighten a 90 degree 3/4&#39; threaded PVC elbow to the male adapter to create the fish tank exhaust pipe. Add a piece of fine mesh before screwing the holding nut to prevent zebrafish going through the exhaust system.</li>
	<li>Place the fish tank with its exhaust pipe ending into the open space of the draining line behind the tanks</li>
</ol>
7. Starting the water system
<ol>
	<li>In several containers, such as the one used for the reservoir, measure three times the amount of water contained within the system. Let this water stand at room temperature for conditioning for 48 hr.</li>
	<li>Add water to the system and let it circulate for a minimum of 3 days.
	<ol>
		<li>Change the water in the system vigorously throughout the initial 3 days of circulation in order to rid the system of contaminants that reside within the pipes.</li>
	</ol>
	</li>
	<li>Add starter zebrafish and allow the nitrogen cycle to get established. Normalize pH to a consistent 7. Keep seven zebrafish per 1.8 L, and feed them twice daily during week days and once daily during weekends as suggested by Linbo4 and McNabb6.</li>
	<li>Monitor the nitrate (&#60;75 mg/L), nitrite (&#60;10 mg/L), and ammonium (0 mg/L) daily during the first week after adding fish, and twice weekly once desired levels have been maintained with regular water changes.</li>
</ol>

<ol>
	<li>Detrich, H., Westerfield, M., Zon, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Zebrafish: Cellular and Developmental Biology, Part A.. , (2010).</li><li>Grim, J. 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=Hemogen+(hemgn),+a+transcription+factor+that+regulates+erythropoiesis+and+skeletogenesis+in+zebrafish.">Hemogen (hemgn), a transcription factor that regulates erythropoiesis and skeletogenesis in zebrafish.</a> , (2013).</li><li>Kim, S., Carlson, R., Zafreen, L., Rajpurohit, S. K., Jagadeeswaran, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular,+easy-to-assemble,+low-cost+facility.">Modular, easy-to-assemble, low-cost facility.</a> Zebrafish. 6 (3), 269-274 (2009).</li><li>Linbo, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+(Danio+rerio)+husbandry+and+colony+maintenance+at+the+Northwest+Fisheries+Science+Center.">Zebrafish (Danio rerio) husbandry and colony maintenance at the Northwest Fisheries Science Center.</a> U.S. Dept. Commer., NOAA Tech. Memo. 62, (2009).</li><li>Avdesh, A., 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=Regular+care+and+maintenance+of+a+zebrafish+(Danio+rerio)+laboratory:+an+introduction.">Regular care and maintenance of a zebrafish (Danio rerio) laboratory: an introduction.</a> J Vis Exp. 69, 4196 (2012).</li><li>McNabb, A., Scott, K., Ochsenstein, E., Seufert, K., Carl, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Don't+be+afraid+to+set+up+your+fish+facility.">Don't be afraid to set up your fish facility.</a> Zebrafish. 9 (3), 120-125 (2012).</li><li>Garcia, R. L., Sanders, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+cleaning+and+disinfection+procedures+in+a+zebrafish+rerio)+facility.">Efficacy of cleaning and disinfection procedures in a zebrafish rerio) facility.</a> J Am Assoc Lab Anim Sci. 50 (6), 895-900 (2011).</li><li>Rivas-Boyero, A. A., 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=Pharmacological+characterization+of+a+nociceptin+receptor+from+zebrafish+(Danio+rerio).">Pharmacological characterization of a nociceptin receptor from zebrafish (Danio rerio).</a> J Mol Endocrinol. 46 (2), 111-123 (2011).</li><li>Sanchez-Simon, F. M., Zhang, X. X., Loh, H. H., Law, P. Y., Rodriguez, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphine+regulates+dopaminergic+neuron+differentiation+via+miR-133b.">Morphine regulates dopaminergic neuron differentiation via miR-133b.</a> Mol Pharmacol. 78 (5), 935-942 (2010).</li><li>Velasco, E. M., Law, P. Y., Rodr&#237;guez, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mu+opioid+from+the+zebrafish+exhibits+functional+characteristics+as+those+of+mammalian+mu+opioid+receptor.">Mu opioid from the zebrafish exhibits functional characteristics as those of mammalian mu opioid receptor.</a> Zebrafish. 6 (3), 259-268 (2009).</li></ol>

The authors declare no conflicts of interest for this paper.

The main purpose of this article is to demonstrate that the construction of a functional zebrafish facility is not complicated and can be built by novice fish researchers with minimal construction knowledge. Furthermore, this article shows that the materials for this functional zebrafish facility can be acquired readily and affordably from local pet supply stores or hardware stores. Once all the materials and tools have been acquired the construction of the system and the fish tanks takes about one working day. The 72 hr...

In vivo models are essential in conducting comprehensive biomedical research since they allow in vitro research to be translated at the system and organismal level. For example, research with small mammals, namely rodents, has been proven necessary in the development of drugs and in the improvement of available drugs. However, the complexity (financially and logistically) might make it virtually impossible to build and maintain rodent facilities for experimentation proposes at small research academic institutions. Nonetheless, in vivo research can be performed using other animal species that require less elaborate logistics. Zebrafish (Danio rerio) have been successfully used as an in vivo model to study genetics, development, pathophysiology, and pharmacology1,2,10,11,12. The facilities required to house zebrafish for experimentation are relatively simple and significantly less expensive when compared to rodent facilities. Kim et al.3 have recently described in detail an 80 tank zebrafish housing system that can be built for approximately $1,500, which includes both parts and labor costs. This stands in stark contrast to the nearly $8,500 that would be required to purchase a ready-made, 80 tank system from any number of commercial suppliers. However, the description given by Kim et al. may be complicated and difficult to follow for novices of fish research. We believe that a zebrafish facility can be constructed in simplified manner for an investment of $1,500.
The establishment of an in vivo facility at small academic institutions provides their faculties an additional tool with which to produce more powerful and thorough research. In vitro data supported by in vivo results generally produce more comprehensive biomedical research than data obtained using only in vitro approaches. Therefore, the implementation of an in vivo research facility will significantly enhance the institution success in its overall research endeavors (student exposure to research, publications, communications, pursuit of external funding, etc.).
The construction of a Zebrafish facility like the one described in this study is ideal for research academic institutions with limited facilities (or institutions without in vivo research facilities) during constricting financial times.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Item</td>
			<td>Brand</td>
			<td>Quantity</td>
			<td></td>
		</tr>
		<tr>
			<td>74-in H x 48-in W x 18-in D 5-shelf Steel Freestanding Shelving Unit</td>
			<td>Style Selections</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>18-Gallon General Bucket/Tote</td>
			<td>Centrex Plastics: LLC&#160;Rugged Tote</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>6-Outlet Power Strip With 15-Ft. Cord</td>
			<td>Master Electrician</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>9-Watt UV Water Garden Clarifier FiIter and UV lamp</td>
			<td>Smartpond&#160;</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>Whole House Water Replacement Filter</td>
			<td>Whirlpool</td>
			<td>6</td>
			<td></td>
		</tr>
		<tr>
			<td>Opaque Whole-House Pre-Filtration Housing</td>
			<td>Whirlpool</td>
			<td>2</td>
			<td></td>
		</tr>
		<tr>
			<td>Thread Seal Tape</td>
			<td>William H Harvey</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>8-fl oz LO-VOC PVC Cement</td>
			<td>Oatey&#160;</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>8-fl oz LO-VOC Primer</td>
			<td>Oatey&#160;</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>1/4-in x 100-ft Vinyl Drip Irrigation Distribution Tubing</td>
			<td>Mister Landscaper&#160;</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>1/4-in Barbed Drip Irrigation On/Off Valve</td>
			<td>Mister Landscaper</td>
			<td>33</td>
			<td></td>
		</tr>
		<tr>
			<td>9-Port NPT Irrigation Manifold with Filter</td>
			<td>&#160;Mister Landscaper</td>
			<td>6</td>
			<td></td>
		</tr>
		<tr>
			<td>Drop-In Filter with House&#160;</td>
			<td>Whirlpool</td>
			<td>6</td>
			<td></td>
		</tr>
		<tr>
			<td>2-in Dia 90-Degree PVC Elbow</td>
			<td>LASCO</td>
			<td>3</td>
			<td></td>
		</tr>
		<tr>
			<td>2-in Dia 90-Degree Tee</td>
			<td>LASCO</td>
			<td>3</td>
			<td></td>
		</tr>
		<tr>
			<td>3/4-in Dia 90-Degree Slip Elbow</td>
			<td>LASCO</td>
			<td>15</td>
			<td></td>
		</tr>
		<tr>
			<td>3/4-in Dia PVC Adapter</td>
			<td>LASCO</td>
			<td>7</td>
			<td></td>
		</tr>
		<tr>
			<td>3/4-in Dia PVC Tee</td>
			<td>LASCO</td>
			<td>12</td>
			<td></td>
		</tr>
		<tr>
			<td>2-in Dia PVC Cap</td>
			<td>LASCO</td>
			<td>3</td>
			<td></td>
		</tr>
		<tr>
			<td>1-in Dia x 3/4-in Dia PVC Adapter</td>
			<td>LASCO</td>
			<td>2</td>
			<td></td>
		</tr>
		<tr>
			<td>3/4-in PVC Socket In-Line Ball Valve</td>
			<td>American Valve</td>
			<td>5</td>
			<td></td>
		</tr>
		<tr>
			<td>1/2-in Dia x 3/4-in Dia PVC Adapter</td>
			<td>Lasco</td>
			<td>8</td>
			<td></td>
		</tr>
		<tr>
			<td>3/4-in Dia PVC Cap</td>
			<td>Lasco</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>2-In x 5-Ft Pvc Pipe</td>
			<td>Charlotte Pipe</td>
			<td>4</td>
			<td></td>
		</tr>
		<tr>
			<td>&#190;-In x 10-Ft Pvc pipe</td>
			<td>Charlotte Pipe</td>
			<td>3</td>
			<td></td>
		</tr>
		<tr>
			<td>11/16x1-7/80 PVC ring</td>
			<td>SXT</td>
			<td>10</td>
			<td></td>
		</tr>
		<tr>
			<td>&#189;&#8221; PVC male adapter</td>
			<td>SXT</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>&#190;&#8221; PVC coupling&#160;</td>
			<td>SXT</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>&#189;&#8221; PVC female adapter&#160;</td>
			<td>SXT</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>link</td>
			<td></td>
		</tr>
		<tr>
			<td>1.8 L Dual Beta Keeper</td>
			<td>Top Fin</td>
			<td>27</td>
			<td></td>
		</tr>
		<tr>
			<td>&#190;&#8221; PVC male adapter (w/ #18 O-ring)</td>
			<td>SXT</td>
			<td>28</td>
			<td></td>
		</tr>
		<tr>
			<td>90-degree &#190;&#8221; threaded PVC elbow</td>
			<td>SXT</td>
			<td>27</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Mesh</td>
			<td>N/A</td>
			<td>1 yard</td>
			<td></td>
		</tr>
		<tr>
			<td>Bulkhead Connector</td>
			<td>N/A</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>Hacksaw</td>
			<td>N/A</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>Drill</td>
			<td>N/A</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>Hole saw</td>
			<td>N/A</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>Adjustable Wrench</td>
			<td>N/A</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>Pliers&#160;</td>
			<td>N/A</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tape Measure</td>
			<td>N/A</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>Safety Glasses</td>
			<td>N/A</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>Work Gloves</td>
			<td>N/A</td>
			<td>1</td>
			<td></td>
		</tr>
	</tbody>
</table>

construction of an affordable and easy-to-build zebrafish facility

In vivo biomedical research is pivotal to translate in vitro findings into clinical advances. Small academic institutions with limited resources find it virtually impossible to build and maintain typical rodent facilities for research. Zebrafish research has been demonstrated to be a valuable alternative for in vivo research in pharmacology, physiology, development and genetic studies. This article demonstrates that a functional zebrafish facility can be built in an easy and affordable manner. We demonstrate that such a facility could be built in about one working day with minimal tools and expertise. The cost of the 27 1.8 L fish tank zebrafish facility constructed in this study was approximately $1,500. We estimate that the maintenance of an initial working 150 fish colony for 3 months is $1,000. This project involved students, who were introduced to aquaculturing of zebrafish for research proposes.

Construction of the Pump and Flters System 
Once the rack is assembled and set up the first step is to build the pump and filter system. The filters are connected to the system pump and are located above the bottom level of the first shelf, which allows for easy filter maintenance and replacement. Similarly, the UV lamp is connected with PVC pipe to the filter and held in place with plastic wire ties to allow for easy replacement of the UV lamp as needed (Figure 4A). Immediately following t...

Watch this Scientific Journal Video about Construction of an Affordable and Easy-to-Build Zebrafish Facility at JoVE.com

Construction of an Affordable and Easy-to-Build Zebrafish Facility

A Zebrafish facility suitable for breeding and in vivo research is built in an easy and affordable manner. Maintenance is minimal. This facility is ideal for student involvement and independent research.

In vivo biomedical research is pivotal to translate in vitro findings into clinical advances. Small academic institutions with limited resources find it ...

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16.2K Views.  Presbyterian College School of Pharmacy. A Zebrafish facility suitable for breeding and in vivo research is built in an easy and affordable manner. Maintenance is minimal. This facility is ideal for student involvement and independent research.

Video: Construction of an Affordable and Easy-to-Build Zebrafish Facility

Proper Care and Cleaning of the Microscope

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;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&#x2019;s role in the regenerating or intact retina.

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and behavioural research. As a vertebrate, zebrafish share considerable genetic sequence similarity with humans and are being used as an animal model for various human disease conditions. The advantages of zebrafish in comparison to other common vertebrate models include high fecundity, low maintenance cost, transparent embryos, and rapid development. Due to the spur of interest in zebrafish research, the need to establish and maintain a productive zebrafish housing facility is also increasing. Although literature is available for the maintenance of a zebrafish laboratory, a concise video protocol is lacking. This video illustrates the protocol for regular housing, feeding, breeding and raising of zebrafish larvae. This process will help researchers to understand the natural behaviour and optimal conditions of zebrafish husbandry and hence troubleshoot experimental issues that originate from the fish husbandry conditions. This protocol will be of immense help to researchers planning to establish a zebrafish laboratory, and also to graduate students who are intending to use zebrafish as an animal model.

Regular Care and Maintenance of a Zebrafish Danio rerio Laboratory: An Introduction

This protocol outlines regular maintenance and care to maintain optimal conditions for zebrafish husbandry. The video illustrates the protocol for system maintenance, regular housing, feeding, breeding, and raising of zebrafish larvae.

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and ...

Microgavage of Zebrafish Larvae

The zebrafish has emerged as a powerful model organism for studying intestinal development1-5, physiology6-11, disease12-16, and host-microbe interactions17-25. Experimental approaches for studying intestinal biology often require the in vivo introduction of selected materials into the lumen of the intestine. In the larval zebrafish model, this is typically accomplished by immersing fish in a solution of the selected material, or by injection through the abdominal wall. Using the immersion method, it is difficult to accurately monitor or control the route or timing of material delivery to the intestine. For this reason, immersion exposure can cause unintended toxicity and other effects on extraintestinal tissues, limiting the potential range of material amounts that can be delivered into the intestine. Also, the amount of material ingested during immersion exposure can vary significantly between individual larvae26. Although these problems are not encountered during direct injection through the abdominal wall, proper injection is difficult and causes tissue damage which could influence experimental results. We introduce a method for microgavage of zebrafish larvae. The goal of this method is to provide a safe, effective, and consistent way to deliver material directly to the lumen of the anterior intestine in larval zebrafish with controlled timing. Microgavage utilizes standard embryo microinjection and stereomicroscopy equipment common to most laboratories that perform zebrafish research. Once fish are properly positioned in methylcellulose, gavage can be performed quickly at a rate of approximately 7-10 fish/ min, and post-gavage survival approaches 100% depending on the gavaged material. We also show that microgavage can permit loading of the intestinal lumen with high concentrations of materials that are lethal to fish when exposed by immersion. To demonstrate the utility of this method, we present a fluorescent dextran microgavage assay that can be used to quantify transit from the intestinal lumen to extraintestinal spaces. This test can be used to verify proper execution of the microgavage procedure, and also provides a novel zebrafish assay to examine intestinal epithelial barrier integrity under different experimental conditions (e.g. genetic manipulation, drug treatment, or exposure to environmental factors). Furthermore, we show how gavage can be used to evaluate intestinal motility by gavaging fluorescent microspheres and monitoring their subsequent transit. Microgavage can be applied to deliver diverse materials such as live microorganisms, secreted microbial factors/toxins, pharmacological agents, and physiological probes. With these capabilities, the larval zebrafish microgavage method has the potential to enhance a broad range of research fields using the zebrafish model system.

We present a novel method for microgavage of larval zebrafish utilizing standard embryo microinjection and stereomicroscopy equipment. We demonstrate that microgavage is a safe and efficient technique useful for delivering controlled amounts of diverse materials specifically into the larval zebrafish intestinal lumen.

The zebrafish has emerged as a powerful model organism for studying intestinal development1-5, physiology6-11, disease12-16, and host-microbe ...

Using an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The presented automatic video tracking system accomplishes this by using a mirror system and a calibration procedure that corrects for the considerable error introduced by the transition of light from water to air. With this system it is possible to record both single and groups of adult zebrafish. Before use, the system has to be calibrated. The system consists of three modules: Recording, Path Reconstruction, and Data Processing. The step-by-step protocols for calibration and using the three modules are presented. Depending on the experimental setup, the system can be used for testing neophobia, white aversion, social cohesion, motor impairments, novel object exploration etc. It is especially promising as a first-step tool to study the effects of drugs or mutations on basic behavioral patterns. The system provides information about vertical and horizontal distribution of the zebrafish, about the xyz-components of kinematic parameters (such as locomotion, velocity, acceleration, and turning angle) and it provides the data necessary to calculate parameters for social cohesions when testing shoals.

The use of a 3D automatic video system that can track individual and groups of zebrafish is described. As application example we explore the effects of the NMDA-receptor antagonist MK-801 on shoals of zebrafish.

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

An Assay for Lateral Line Regeneration in Adult Zebrafish

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.17&#160;that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish rather than the embryo/larvae, we developed a quantitative lateral line regenerative assay that can be applied to adult zebrafish disease models. The assay involved resolution at the 1) neuromast and 2) individual hair cell levels.

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a &#8216;targeting&#8217; vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term &#8216;subcloning plus insertion&#8217;, which can be used to generate custom gene targeting vectors rapidly.

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic ...