We thank members of the Miller lab for discussion and critical reading of the manuscript. This work was supported by a new investigator award from the Nathan Shock Center of Excellence in the Basic Biology of Aging to DLM and the National Institutes of Health award R00 AG030550 to DLM.

1. Construction of Environmental Chambers
<ol>
	<li>Select the smallest reasonable volume of chamber required for the scope of your project. Chamber must be made of gas (O2) impermeable material. Pyrex crystallization dishes, Anaeropack boxes, or large cast-acrylic boxes (Ellard Instrumentation), can be used. We have found that 9 50 mm plates can fit in a 100 x 50 Kimex crystallization dish. Glass plates can be used as lids for Pyrex crystallization dishes.</li>
	<li>Drill a hole in the selected chamber and fit with a plastic male Luer to hose barb fitting (Cole Parmer). Fittings can be secured by pipe fitting or with epoxy. Install a similar fitting on the opposite side of the container to allow for gas to flow in and out of the chamber. If possible, offset holes to increase turbulent mixing.</li>
	<li>Obtain compressed gas tanks with defined O2 concentrations (balanced with N2) that are certified standard for O2 content or, for anoxic conditions, pure N2 (&lt;10 ppm O2). Use automatic switch-over regulators for longer-term studies to avoid disrupting the oxygen levels in the chambers.</li>
	<li>Organismal response to hypoxia has been shown to be temperature dependent15. By placing the chamber in an incubator, different temperatures can be maintained. Temperatures within an incubator may be uneven and as such, it is prudent to make use of a temperature data logger to constantly measure the temperature inside the chamber.</li>
</ol>
2. Connecting the Gas to the Environmental Chamber
<ol>
	<li>For all connections, use one-eighth-inch outer-diameter tubing connected by either snap connectors or compression fittings. Tubing should be impermeable and unreactive with O2, such as fluorinated ethylene propylene (FEP) or nylon (Cole Parmer). For a schematic of the completed setup, see Figure 1.</li>
	<li>Connect the compressed gas tanks to a flow control device, such as a mass flow controller (Sierra Instruments) or rotameter (Aalborg). Ensure that upstream pressure from the tank is within the range of the flow device and the hose barb fittings. Two-stage regulators are generally used, with the second stage set to the desired pressure [See section three for selecting the appropriate flow rate]</li>
	<li>Hydrate the gas by bubbling through distilled water using a gas wash bottle with fritted cylinder, then direct into one of the fittings on the environmental chamber, leaving the second fitting open for gas exhaust (see Figure 1). For short-term studies, gas hydration protects against plate dessication, but humidity monitoring may be necessary for long-term studies.</li>
	<li>Dow Corning Vacuum Grease can be used to seal the chamber. Place weights on the lid of the chamber to ensure an airtight seal. To confirm a tight seal and adequate flow, hold a small pool of water in the palm of your gloved hand to the out fitting on the chamber and check for bubbles.</li>
</ol>
3. Selecting Flow Rate
<ol>
	<li>Assuming perfect mixing, there is 90% gas exchange of the gaseous atmosphere each time the volume of the chamber is replaced (Fick's Law). For example, in a 100 cc chamber with a flow rate of 100 cc/min, the original house air in the chamber will be replaced with 90% of your desired gas after 1 minute, and will asymptotically approach complete exchange by 90% every minute thereafter.</li>
	<li>Higher flow rates and smaller containers will reach your desired oxygen concentration more quickly. For 100 x 50 Kimex containers (400 cc), a flow rate of 120 cc/min will reach 99.9% exchange in 10 minutes (3 exchanges). This flow rate is suitable for most oxygen conditions. To our knowledge there has not been a systematic investigation of how the rate of change of O2 concentration influences the response in C. elegans.</li>
</ol>
4. Preparation of Samples for Viability Assay
<ol>
	<li>Worms exposed to hypoxic conditions commonly escape the surface of agar plates. To prevent this, place a ring of palmitic acid (10 mg/ml in ethanol) around the edge of the plates. The palmitic acid will come out of solution as the ethanol evaporates, forming a physical barrier. Palmitic acid barriers do not affect rate of egg laying, fecundity or lifespan in C. elegans16. Burrowing does not occur more frequently in hypoxic conditions, so additional preventative measures are not generally required.</li>
	<li>
	<ol>
		<li>Generate synchronized populations by bleaching gravid adults in a small drop of alkaline bleach solution on unseeded nematode growth media (NGM) plates17. In contrast to standard large-batch hypochlorite bleaching protocols, pick 1-100 animals in a drop of bleach solution on the surface of an NGM plate, then allow the bleach solution to absorb into the plate17. After at least 12 hours, transfer the synchronized L1 larvae to plates seeded with live OP50 bacteria. Alternatively, one can allow gravid adults to lay eggs on the plate for 2-3 hours to generate a group of worms that will develop synchronously or pick L4 larvae from a mixed population.</li>
		<li>Avoid exposing bleached embryos to hypoxia, as this can reduce viability13. To collect young embryos (2-4 cells), gravid adults can be chopped in a small volume of water with a razor blade and embryos moved to plates by mouth pipet for subsequent exposure to hypoxia.</li>
	</ol>
	</li>
	<li>Seal plates in the environmental chamber. Control animals should be kept in normoxia (house air) at the same temperature as treated worms. There is no observable difference between samples left in house air and those maintained in an identical chamber with house air flowing over them. Initiate gas flow and maintain exposure for desired time. To ensure uniformity in ramp, be sure to replace the water in the gas wash bottle before exposure.</li>
	<li>To assay survival of embryos, allow the worms to develop for 48 h after return to room air, at which point they should be fourth-stage larvae/day one adults. Score for survival, censoring any worms that cannot be accounted for.</li>
	<li>To visualize animals exposed to hypoxia, move worms to a drop of M9 on a 22 mm2 coverslip, and invert onto a pad of 2% agarose in M917. If necessary, levamisole (25 mM) or sodium azide (10 mM) can be used as anesthetic. Sodium azide and levamisole may confound some observations due to toxicity and should be judiciously used18.</li>
</ol>
5. Rapid Harvest of Hypoxia-exposed Worms (Example: Preparation of Samples for HIF-1 Western Analysis)
Many hypoxia-induced effects are quickly reversed upon return to room air, including the resumption of egg production12, phosphorylation of mitotic epitopes in embryogenesis13 and degradation of the HIF-1 protein9,19. Rapid isolation of animals exposed to hypoxia is required to obtain reproducible effects in these conditions. With this setup, animals can be harvested and frozen in liquid nitrogen in less than two minutes. While glove box hypoxia chambers allow for manipulation of samples in anoxic conditions, their cost and practicality for conditions other than anoxia limit their usefulness.
<ol>
	<li>Grow Bristol N2 worms on 4 10 cm high growth (HG) plates until a majority of the worms are gravid adults17. Wash worms to a 15 mL conical tube containing a 1:5 alkaline bleach solution and incubate with rotation until worms begin to dissolve, not more than 5 minutes9. Wash the worms three times with M9, spinning down at 1500 x g between each wash with no braking.</li>
	<li>Plate bleached embryos onto 8 150 mm NGM plates and allow to develop to L4 larvae (~48 hours for Bristol N2 at 22 &deg;C). Move plates to environmental chambers and expose to hypoxic (1,000 ppm, 5,000 ppm) and anoxic (N2) conditions for 4 hours. Exposure times will vary depending on experimental design. While exposure to hypoxia has an immediate effect on rate of egg laying, two cell embryos die after 16-18 hours of exposure12. With this hypoxia chamber setup, the lower limit of exposure is constrained by the rate of atmosphere exchange necessary to reach equilibrium.</li>
	<li>Label one 1.5 mL microfuge tube and one 15 mL conical tube for each experimental sample. Worms exposed to hypoxia are more likely to stick to the sides of the tube during harvesting. To prevent this, place 100 &mu;L of 1% sodium dodecyl sulfate (SDS) in each 15 mL conical tube. If SDS inhibits downstream applications, bovine serum albumin (BSA) can be used to prevent sticking. Routine use of SDS or BSA does not seem to have an apparent difference. Add 50 &mu;l of 2x protein loading dye (4% SDS, 10% 2-Mercaptoethanol and a trace of bromphenol blue in 30% glycerol (w/v)) to the 1.5 mL microfuge tube. Have a Dewar of liquid nitrogen ready.</li>
	<li>Time the steps after removing the worms from hypoxia and record. Remove the lid to the hypoxic chamber, take one sample plate, and reseal the chamber. Use distilled water to wash the worms onto a nylon filter and then pour into the 15 mL conical tube. Spin the worms down in a desktop centrifuge at 1500 x g for 15-20 seconds with brake.</li>
	<li>Use a vacuum to remove most of the supernatant from the tube, leaving the worm pellet untouched.</li>
	<li>Using a pipette, move the worm pellet in 50 &mu;L to the 1.5 mL microfuge tube. Seal the tube and immerse in liquid nitrogen.</li>
	<li>Repeat until all samples have been isolated. Follow these procedures for house air control samples for consistency. Samples can be stored at -20 &deg;C.</li>
</ol>
6. Representative Results
Organismal effects of hypoxia can be seen by examining the viability to adulthood of C. elegans (Figure 2). Embryos laid by wild-type Bristol (N2) and hif-1(ia04) deletion mutants are all survive in house air O2 concentrations (210,000 ppm O2). N2 worms are able to adapt and survive to adulthood in 5,000 ppm O2, while hif-1 embryos are not viable. This shows that HIF-1 is essential for adapting to the changing levels of oxygen available in the environment10. Neither N2 nor hif-1 animals can survive exposure to 1,000 ppm O2.
Visualizing worm directly in hypoxia is feasible with the use of a dissecting scope and clear container (Figure 3). By directly placing the hypoxia chamber on the dissecting scope, there is no need to remove the worms from hypoxia to observe organismal reactions. The scope can be fitted with fluorescence illumation (as in Figure 3), further extending the types of observations in hypoxia that are possible.
<img alt="Figure 1" src="/files/ftp_upload/4088/4088fig1.jpg" /> 
Figure 1. Example of Hypoxia chamber. Direction of gas flow is indicated by arrows. Gas is stored in compressed gas tanks with defined O2 concentrations (1) and a two stage regulator is attached (2). Gas enters the bottom of the flow tube (3), exiting the top at the correct flow rate. Gas then flows into the bubble flask (4), hydrating the gas (ensure correct connection of bubble flask by observing bubbles). Hydrated gas then passes into the hypoxia chamber at the inflow valve (5), exposing the samples to hypoxia. The gas finally vents into the room through an exhaust hole drilled in the chamber.
<img alt="Figure 2" src="/files/ftp_upload/4088/4088fig2.jpg" /> 
Figure 2. Viability of embryos exposed to 1,000 ppm O2, 5,000 ppm O2 and normoxia (~210,000 ppm O2). Embryos were exposed to each oxygen conditions as embryos for 24 hours in continuous flow oxygen chambers. Worms were moved to normoxic conditions, allowed to develop to adulthood for 48 hours, and then scored for viability to adulthood. n&gt;50, N=5.
<img alt="Figure 3" src="/files/ftp_upload/4088/4088fig3.jpg" /> 
Figure 3. Visualization of C. elegans in hypoxia with microscopy. Worms are exposed to hypoxia using the methods outlined. The transparent environmental chamber (constructed with a pyrex crystallization dish and glass plate) is placed directly on the stage of a dissecting scope. Two views are shown, one including the entire gas flow set up, the other with just the chamber on the microscope stage.

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No conflicts of interest declared.

This method presents a strategy for constructing a hypoxic environment that allows for environments with precise concentrations of oxygen to be maintained in the laboratory. These chambers provide a simple method for exposing organisms to specific low concentrations of O2 and monitoring the molecular and physiological outputs. The environmental chamber described is assembled by the lab instead of commercially purchased and can thus be modified to fit the needs of the experiment.
One...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Tubing (FEP and PTFE)</td>
			<td>Cole Parmer 
			Tygon</td>
			<td>YO-95821-00 (1/8" FEP) 
			06605-27 (1/16 x 1/8" PTFE)' R-3603</td>
		</tr>
		<tr>
			<td>Compression fittings</td>
			<td>Seattle Fluid Systems</td>
			<td>06363-58 (M. coupler 1/16") 
			06363-62 (F. coupler 1/16") 
			06363-60 (M. coupler 1/8") 
			06363-61 (F. coupler 1/8")</td>
		</tr>
		<tr>
			<td>Flow tube</td>
			<td>Aalborg</td>
			<td>PMR3-010073 (3 output) 
			PMR1-013520 (1 output)</td>
		</tr>
		<tr>
			<td>Mass flow controller</td>
			<td>Sierra Instruments</td>
			<td>810S-L-DR-2-OV1-SK1-V1-S1 (Mass Trak) 
			C100L-DD-2-OV1-SV1-PV2-V1-SO-C10 (Smart Trak 2)</td>
		</tr>
		<tr>
			<td>Compressed gas tank</td>
			<td>AirGas</td>
			<td>Made to order</td>
		</tr>
		<tr>
			<td>Plastic male Luer to hose barb fittings</td>
			<td>Cole Parmer</td>
			<td>45505-41 (500 series 1/16")</td>
		</tr>
		<tr>
			<td>Cast acrylic boxes</td>
			<td>Ellard Instrumentation</td>
			<td>Made to order</td>
		</tr>
		<tr>
			<td>Pipe fittings (Brass or stainless steel)</td>
			<td>Seattle Fluid Systems</td>
			<td>B-402-1 (1/4" nut) 
			B-200-3 (1/8" union tee) 
			B-400-set (1/4" ferrules) 
			B-QM2-B1-200 (QM Body QC) 
			B-200-1-2 (1/8 x 1/8" male conn)</td>
		</tr>
		<tr>
			<td>Dow Corning Vacuum Grease</td>
			<td>Sigma-Aldrich</td>
			<td>Z273554</td>
		</tr>
		<tr>
			<td>AnaeroPack box</td>
			<td>Misubishi Gas Chemical Company</td>
			<td>R684004 (0.4 liter) 
			R685025 (2.5 liter) 
			R685070 (7.0 liter)</td>
		</tr>
		<tr>
			<td>Pyrex gas wash bottle</td>
			<td>Sigma-Aldrich</td>
			<td>CLS31770500C (500 mL) 
			CLS31770250C (250 mL) 
			CLS31770125C (125 mL)</td>
		</tr>
		<tr>
			<td>Palmitic acid</td>
			<td>Sigma-Aldrich</td>
			<td>P0500</td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG-horseradish peroxidase</td>
			<td>Southern Biotechnology Associates</td>
			<td>1032-05</td>
		</tr>
		<tr>
			<td>SuperSignal West Pico Chemiluminsecent Substrate</td>
			<td>Pierce Chemical</td>
			<td>34077</td>
		</tr>
		<tr>
			<td>100 x 50 glass crystallization dishes</td>
			<td>Kimax Kimble</td>
			<td>23000</td>
		</tr>
	</tbody>
</table>

creating defined gaseous environments to study the effects of hypoxia on c. elegans

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, normal development or changes in environmental conditions2-5. Understanding the cellular signaling pathways that are involved in the response to hypoxia could provide new insight into treatment strategies for diverse human pathologies, from stroke to cancer. This goal has been impeded, at least in part, by technical difficulties associated with controlled hypoxic exposure in genetically amenable model organisms.
The nematode Caenorhabditis elegans is ideally suited as a model organism for the study of hypoxic response, as it is easy to culture and genetically manipulate. Moreover, it is possible to study cellular responses to specific hypoxic O2 concentrations without confounding effects since C. elegans obtain O2 (and other gasses) by diffusion, as opposed to a facilitated respiratory system6. Factors known to be involved in the response to hypoxia are conserved in C. elegans. The actual response to hypoxia depends on the specific concentration of O2 that is available. In C. elegans, exposure to moderate hypoxia elicits a transcriptional response mediated largely by hif-1, the highly-conserved hypoxia-inducible transcription factor6-9. C .elegans embryos require hif-1 to survive in 5,000-20,000 ppm O27,10. Hypoxia is a general term for "less than normal O2". Normoxia (normal O2) can also be difficult to define. We generally consider room air, which is 210,000 ppm O2 to be normoxia. However, it has been shown that C. elegans has a behavioral preference for O2 concentrations from 5-12% (50,000-120,000 ppm O2)11. In larvae and adults, hif-1 acts to prevent hypoxia-induced diapause in 5,000 ppm O212. However, hif-1 does not play a role in the response to lower concentrations of O2 (anoxia, operational definition &lt;10 ppm O2)13. In anoxia, C. elegans enters into a reversible state of suspended animation in which all microscopically observable activity ceases10. The fact that different physiological responses occur in different conditions highlights the importance of having experimental control over the hypoxic concentration of O2.
Here, we present a method for the construction and implementation of environmental chambers that produce reliable and reproducible hypoxic conditions with defined concentrations of O2. The continual flow method ensures rapid equilibration of the chamber and increases the stability of the system. Additionally, the transparency and accessibility of the chambers allow for direct visualization of animals being exposed to hypoxia. We further demonstrate an effective method of harvesting C. elegans samples rapidly after exposure to hypoxia, which is necessary to observe many of the rapidly-reversed changes that occur in hypoxia10,14. This method provides a basic foundation that can be easily modified for individual laboratory needs, including different model systems and a variety of gasses.

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Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

This paper details how to use continuous-flow hypoxia chambers to generate atmospheres with defined concentrations of O2 to understand biological responses to decreased O2. This system is easy to setup and maintain, and flexible enough to suit a wide range of O2 concentrations and model systems

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, ...

creating-defined-gaseous-environments-to-study-effects-hypoxia-on-c

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JoVE Journal

Biology

12.0K Views.  University of Washington. В настоящем документе подробно описано, как использовать непрерывный поток гипоксии камеры для создания атмосферы с определенной концентрации O 2, Чтобы понять биологические реакции со снижением O 2. Эта система проста в установке и обслужив...

Video: Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. 
The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. 
The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and expression pattern of genes of interest or generation of tandem affinity purification (TAP) tagged versions of specific genes to facilitate their purification. Typically transgenes are generated by placing a promoter upstream of a GFP reporter gene or cDNA of interest, and this often produces a representative expression pattern. However, critical elements of gene regulation, such as control elements in the 3' untranslated region or alternative promoters, could be missed by this approach. Further only a single splice variant can be usually studied by this means. In contrast, the use of worm genomic DNA carried by fosmid DNA clones likely includes most if not all elements involved in gene regulation in vivo which permits the greater ability to capture the genuine expression pattern and timing. To facilitate the generation of transgenes using fosmid DNA, we describe an E. coli based recombineering procedure to insert GFP, a TAP-tag, or other sequences of interest into any location in the gene. The procedure uses the galK gene as the selection marker for both the positive and negative selection steps in recombineering which results in obtaining the desired modification with high efficiency. Further, plasmids containing the galK gene flanked by homology arms to commonly used GFP and TAP fusion genes are available which reduce the cost of oligos by 50% when generating a GFP or TAP fusion protein. These plasmids use the R6K replication origin which precludes the need for extensive PCR product purification. Finally, we also demonstrate a technique to integrate the unc-119 marker on to the fosmid backbone which allows the fosmid to be directly injected or bombarded into worms to generate transgenic animals. This video demonstrates the procedures involved in generating a transgene via recombineering using this method.

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...