The authors would like to thank Drs. Roger Cone and Chao Zhang for their contributions to validating the application of this assay as previously published. This work was supported by NIH 1F32DK082167-01 (BJR).

Note: This protocol follows the guidelines of the University of Arizona Office for the Responsible Conduct of Research and has been approved by the Institutional Animal Care and Use Committee
1. Zebrafish Breeding and Embryo Maintenance
<ol>
	<li>Prepare E3 embryo medium8. To Make 60x stock solution, dissolve 34.8 g NaCl, 1.6 g&#160;KCl, 5.8g CaCl2&#9679;2H2O, and 9.78 g MgCl2&#9679;6H2O in 1.95 L ddiH2O....

<ol>
	<li>Jao, L. E., Wente, S. R., Chen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+multiplex+biallelic+zebrafish+genome+editing+using+a+CRISPR+nuclease+system.">Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system.</a> PNAS. 110, 13904-13909 (2013).</li><li>Kimura, Y., Hisano, Y., Kawahara, A., Higashijima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+generation+of+knock-in+transgenic+zebrafish+carrying+reporter/driver+genes+by+CRISPR/Cas9-mediated+genome+engineering.">Efficient generation of knock-in transgenic zebrafish carrying reporter/driver genes by CRISPR/Cas9-mediated genome engineering.</a> Sci. Rep. 4, 6545 (2014).</li><li>Lieschke, G. J., Currie, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+human+disease:+zebrafish+swim+into+view.">Animal models of human disease: zebrafish swim into view.</a> Nat. Rev. Genet. 8, 353-367 (2007).</li><li>Brand, M. D., Couture, P., Else, P. L., Withers, K. W., Hulbert, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+energy+metabolism.+Proton+permeability+of+the+inner+membrane+of+liver+mitochondria+is+greater+in+a+mammal+than+in+a+reptile.">Evolution of energy metabolism. Proton permeability of the inner membrane of liver mitochondria is greater in a mammal than in a reptile.</a> Biochem. J. 275 (Pt 1), 81-86 (1991).</li><li>Renquist, B. J., Zhang, C., Williams, S. Y., Cone, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+assay+for+high-throughput+energy+expenditure+monitoring+in+the+zebrafish.">Development of an assay for high-throughput energy expenditure monitoring in the zebrafish.</a> Zebrafish. 10, 343-352 (2013).</li><li>Livingston, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+volumetric+respirometer+for+long-term+studies+of+small+aquatic+animals.">A volumetric respirometer for long-term studies of small aquatic animals.</a> J.Mar. Biol. Assoc. U.K. 48, 485-497 (1968).</li><li>Makky, K., Duvnjak, P., Pramanik, K., Ramchandran, R., Mayer, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+whole-animal+microplate+assay+for+metabolic+rate+using+zebrafish.">A whole-animal microplate assay for metabolic rate using zebrafish.</a> J. Biomol. Screen. 13, 960-967 (2008).</li><li>Nasiadka, A., Clark, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+breeding+in+the+laboratory+environment.">Zebrafish breeding in the laboratory environment.</a> ILAR J. 53, 161-168 (2012).</li><li>Brand, M., Granato, M., Nusslein-Volhard, C., Nusslein-Volhard, C., Dahm, R. <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. , (2002).</li><li>Fry, F. E. J., Hart, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cruising+speed+of+goldfish+in+relation+to+water+temperature.">Cruising speed of goldfish in relation to water temperature.</a> J. Fish. Res. Board Can. 7, 169-175 (1948).</li><li>Oka, T., 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=Diet-induced+obesity+in+zebrafish+shares+common+pathophysiological+pathways+with+mammalian+obesity.">Diet-induced obesity in zebrafish shares common pathophysiological pathways with mammalian obesity.</a> BMC physiol. 10 (21), (2010).</li><li>Song, Y., Cone, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creation+of+a+genetic+model+of+obesity+in+a+teleost.">Creation of a genetic model of obesity in a teleost.</a> FASEB J. 21, 2042-2049 (2007).</li><li>Maddison, L. A., Joest, K. E., Kammeyer, R. M., Chen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+insulin+resistance+in+zebrafish+induces+alterations+in+beta-cell+number+and+glucose+tolerance+in+an+age+and+diet+dependent+manner.">Skeletal muscle insulin resistance in zebrafish induces alterations in beta-cell number and glucose tolerance in an age and diet dependent manner.</a> Am. J. physiol. Endocrinol. , (2015).</li><li>Flynn, E. J., Trent , C. M., Rawls, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ontogeny+and+nutritional+control+of+adipogenesis+in+zebrafish+(Danio+rerio).">Ontogeny and nutritional control of adipogenesis in zebrafish (Danio rerio).</a> J. lipid res. 50, 1641-1652 (2009).</li><li>Shimada, Y., Hirano, M., Nishimura, Y., Tanaka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+fluorescence-based+assay+system+for+appetite-regulating+gene+and+drug+screening.">A high-throughput fluorescence-based assay system for appetite-regulating gene and drug screening.</a> PloS one. 7, e52549 (2012).</li><li>Tingaud-Sequeira, A., Ouadah, N., Babin, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+obesogenic+test:+a+tool+for+screening+molecules+that+target+adiposity.">Zebrafish obesogenic test: a tool for screening molecules that target adiposity.</a> J. lipid res. 52, 1765-1772 (2011).</li></ol>

Contamination with bacteria or fungi will greatly limit application of this assay. The methylene blue in the E3 embryo medium limits the possibility of fungal contamination. Throughout embryo rearing it is critical that care is taken to remove dead embryos twice each day. Additionally, it is essential to thoroughly rinse the embryos with sterile E3 embryo medium on the day of assay initiation. These 2 steps limit the potential for bacterial contamination of the embryos. The inclusion of blank wells described in step 3.2 ...

The mouse is currently the predominant model for obesity research. The short generation interval and genetic tools available in the mouse have been unmatched to date. However, the zebrafish also has a short generation interval (3 - 4 months) and surpasses even the mouse in ease of genetic manipulation 1,2. The zebrafish maintains nearly 90% of mammalian genes, while far exceeding the mouse in number of offspring and potential for use in genetic and drug screens 3.
To tap the potential of the zebrafish model for studies in obesity, assays must be developed to investigate factors that influence body weight regulation, including energy expenditure. As metabolites are processed through &#946;-oxidation and the tricarboxylic acid cycle oxygen is consumed and NADH2 is produced. Thus, NADH2 is a direct indicator of the flux of metabolites through metabolic pathways (metabolite oxidation). In poikilotherms, H+ leak through the inner mitochondrial membrane, which uncouples NADH2 oxidation from ATP synthesis, is 4 - 5 fold lower than in homeotherms 4. Accordingly, in zebrafish NADH2 is very tightly linked to ATP production through oxidative phosphorylation. Herein, we describe an assay that measures NADH2 production in larval zebrafish as a proxy for energy expenditure5.
Oxygen consumption is the gold standard for measuring energy expenditure. Yet, to best take advantage of the high throughput potential of the zebrafish, assays of energy expenditure must be amenable to high-throughput. Oxygen consumption systems that depend on a closed chamber circulating system are limited in throughput by the number of chambers available6. Open air O2 consumption/CO2 production assays have also been applied in the zebrafish5,7. These open air 96-well plate based systems are amenable to high throughput. Unfortunately, gas exchange with the environment limits sensitivity of these assays. We recently published the application of an assay that monitors NADH2 using the redox indicator alamarBlue5. This assay overcomes the limitations in throughput and sensitivity common to analyses of oxygen consumption in the zebrafish.
The zebrafish is becoming an increasingly important model for studies of whole body energy homeostasis. In part, because zebrafish are amenable to use in forward genetic screens and drug screens. Moreover, targeted genetic manipulation, including knockdown and knock-in, can be quickly applied. We have previously shown that this assay can be combined with bath drug application and genetic knockdown or knockout to identify compounds and genes that alter metabolic rate5. Moreover, this assay is designed to exploit the high throughput advantages inherent to the zebrafish.

<table border="0" cellpadding="0" cellspacing="0" width="966">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">AlamarBlue</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">10-230-103</td>
			<td style="width:307px;">Manufactured by Thermo Scientific</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Fine Tip DisposableTransfer Pipette</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td>13-711-26</td>
			<td>Manufactured by Thermo Scientific</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Fisherbrand Graduated Disposable Transfer Pipette</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td>13-771-9AM</td>
			<td>Manufactured by Thermo Scientific</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Black sided clear bottom 96-well plates</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">50-320-785</td>
			<td>Manufactured by E&#38;K Scientific Products Inc.</td>
		</tr>
	</tbody>
</table>

high throughput danio rerio energy expenditure assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).

Assay solution does not increase fluorescence in the absence of embryonic fish (Figure 1). However, the assay is highly sensitive to small changes in metabolic rate within the well. A single fish within a well creates a signal significantly different from blank wells within 1 hr (P&#60;0.0001). Figure 2 provides a visual representation of signals generated by embryonic fish after 24 hr of incubation. For the purpose of this picture clear sided wells were used. Yet, black sided wells are ...

Watch this Scientific Journal Video about High Throughput Danio Rerio Energy Expenditure Assay at JoVE.com

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...

The overall goal of this high throughput resazurin-based fluorometric assay is to monitor the effect of genetic or pharmacological manipulations on NADH2 production, which is a measure of metabolic rate. This method can help us answer key questions in the obesity field, such as the affect of a given drug or gene on metabolic rate. Additionally, this assay can be applied to screen for potential drug-drug or drug-gene interactions in a whole animal system.The main advantage of this assay, relative to oxygen consumption assays, is the high throughput and the accumulation of signal across time, which allows the user to detect small differences in metabolic rate. Demonstrating this procedure will be Caroline Foy, a graduate student in my laboratory. After preparing a 60X stock solution of E3 medium according to the text protocol, prepare 1X E3 medium by diluting 16.5 milliliters of stock and one liter of double distilled water.Then add 100 microliters of 1%methylene blue. To make flame polish disposable pipette for egg and fish transfer, use scissors to cut a graduated disposable transfer pipette at the 0.25 milliliter graduation. Then, to remove the rough edges, use a Bunsen burner to flame polish the cut tip.After setting up zebrafish crossings and collecting eggs, allow the eggs to settle to the bottom of the dish, and pour off the water. Use a fine-tip disposable transfer pipette to remove any remaining water. Then add back the E3 medium.Incubate the embryonic fish at 28.5 degrees Celsius to four days post fertilization, or DPF. Twice each day use a flame polished graduated disposable transfer pipette to remove any unfertilized eggs or dead embryos. Then once each day pour off the E3 medium.Use a fine-tip transfer pipette to remove any remaining medium, and replace with fresh E3 pre-warmed to 28.5 degrees Celsius. To carry out the energy expenditure assay, prepare the assay solution, following this table. Use a 0.22 micrometer filter to filter sterilize the solution, and warm to 28.5 degrees Celsius in a water bath for the assay.In a petri dish, combine all viable zebrafish embryos from the clutch. After filter sterilizing 75 milliliters of E3 medium, use a fine-tip disposable transfer pipette to remove as much of the E3 medium from the petri dish as possible. Add back 20 milliliters of sterile E3.Then remove and replace the sterile E3 two more times.Next, using a flame-polished graduated plastic disposable transfer pipette, transfer individual embryonic fish to the wells of a 96-well plate. Working with one column of the plate at a time, remove the E3 medium from the eight wells of the first column, taking care not to touch the embryos with the transfer pipette. Then add 300 microliters of assay solution to each well.Repeat with all 12 columns of the plate. To include a drug treatment, after preparing a 100X solution of the desired compound, add three microliters to each of the treatment wells. Then add three microliters of vehicle control to the control wells of fish.To ensure that the fluorescence response to the compound is a result of activity within the fish, set up drug and vehicle treatments to three blank wells each in a 96-well plate. Now, using a fluorescence plate reader, with excitation and emission wave lengths of 530 nanometers and 590 nanometers respectively, read the wells of the fish plate. Place the plate into a humidified incubator at 28.5 degrees Celsius.Depending on the assay and the stability of the compound being tested, read the fluorescence again at a predetermined time point. To assess the relative change in fluorescence, calculate the average change in fluorescence for the control wells. Then divide the change in fluorescence of each well by the average change in fluorescence of the control wells.Here is an example of using two columns of a 96-well plate. Column A wells include vehicle-treated control zebrafish, while column B wells include insulin-treated zebrafish. In table A, the fluorescence measured at time zero is shown.Table B contains the fluorescence measured 24 hours later. In table C, the fluorescence at time zero has been subtracted from the fluorescence at 24 hours to assess the change in fluorescence. Next, calculate the average change in fluorescence in control wells.Table D is the change in fluorescence of each individual well, divided by the average change in the control wells. You can see here that insulin treatment at 10 micromolar caused a 2.77 fold increase in the signal generated by zebrafish over 24 hours by a P value of less than 0.0001. As seen here, the assay solution does not change color or absolute fluorescence levels in the absence of embryos.However, the assay is highly sensitive to small changes in metabolic rate within the well. This figure represents signals generated by embryonic tilapia after incubating for 24 hours. Pink wells are indicative of fish with a high metabolic rate.Purple wells contain fish with moderate metabolic rates, and the blue wells represent a low metabolic rate. This also shows the versatility of this assay and application across teleost species. Because the NADH2 induced reduction of alamar blue is non-reversible, the signal accumulates with time, which allows for small changes to be amplified.Shown here is the relative change in fluorescence induced by one to five fish at one, two and four hours. With each increase in the number of fish, the relative change in fluorescence increase significantly with time, and the greater the number of fish, the greater the magnitude in fluorescence change with time. Once mastered, this technique can be performed on up to 5, 000 embryonic fish per day.While attempting this procedure, it's important to remember to take care to avoid injuring the embryonic fish. After performing this assay, other procedures like Nile red measurement of neutral lipids, or measurement of phagic drive through intake of fluorassay labeled paramecium can be performed. This allows us to compare the relationship between metabolic rate and lipid metabolism, or metabolic rate and phagic drive.After its development, this technique paved the way for researchers in the field of energy metabolism and metabolic disease to study energy expenditure in zebrafish without limitations in throughput. After watching this video, you should have a good understanding of how to use the fluorometric reagent resazurin to measure NADH2 production in the zebrafish.

high-throughput-danio-rerio-energy-expenditure-assay

Research

JoVE Journal

Biology

12.5K vues.  Vanderbilt University Medical Center. The overall goal of this high throughput resazurin-based fluorometric assay is to monitor the effect of genetic or pharmacological manipulations on NADH2 production, which is a measure of metabolic rate. This method can help us answer key questions in the obesity field, such as the affect of a given drug or gene on metabolic rate. Additionally, this assay can be applied to screen for potential drug-drug or drug-gene interactions in a whole animal system.The main advantage of this assay...

Vidéo: High Throughput Danio Rerio Energy Expenditure Assay

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss.
Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish Danio rerio

The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the ...

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

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 ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

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.

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...