Name: scalable, flexible, and cost-effective seedling grafting
Uploaded: 2023-01-06T13:20:00
Description: Watch this Scientific Journal Video about Scalable, Flexible, and Cost-Effective Seedling Grafting at JoVE.com

Thanks to Javier Brumos for initial training and guidance in grafting Arabidopsis seedlings.

1. Device preparation
<ol>
	<li>Make the silicone grafting device by casting silicone elastomer solution in a square Petri dish (100 mm x 100 mm). Prepare 15 mL of the elastomer solution, following the manufacturer&#39;s guidelines. 
	NOTE: Silicone elastomer kits typically include a silicone-based liquid and a curing agent, that when mixed together allow the silicone to solidify.</li>
	<li>Prepare the square Petri dish by laying four straight pieces of 29 G wire in the square Petri dish, equidist...

<ol>
	<li>Mudge, K., Janick, J., Scofield, S., Goldschmidt, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+history+of+grafting.">A history of grafting.</a> Horticultural Reviews. 35, 437-493 (2009).</li><li>Holbrook, N. M., Shashidhar, V. R., James, R. A., Munns, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stomatal+control+in+tomato+with+ABA-deficient+roots:+Response+of+grafted+plants+to+soil+drying.">Stomatal control in tomato with ABA-deficient roots: Response of grafted plants to soil drying.</a> Journal of Experimental Botany. 53 (373), 1503-1514 (2002).</li><li>Notaguchi, M., Okamoto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+long-distance+signaling+via+plant+vascular+tissues.">Dynamics of long-distance signaling via plant vascular tissues.</a> Frontiers in Plant Science. 6, 161 (2015).</li><li>Ko, D., Helariutta, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shoot-root+communication+in+flowering+plants.">Shoot-root communication in flowering plants.</a> Current Biology. 27 (17), 973-978 (2017).</li><li>Thomas, H. R., Frank, M. 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G. N., Booker, J. P., Leyser, H. M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micrografting+techniques+for+testing+long-distance+signalling.">Micrografting techniques for testing long-distance signalling.</a> The Plant Journal. 32 (2), 255-262 (2002).</li><li>Marsch-Mart&#237;nez, N., 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=An+efficient+flat-surface+collar-free+grafting+method+for+Arabidopsis+thaliana+seedlings.">An efficient flat-surface collar-free grafting method for Arabidopsis thaliana seedlings.</a> Plant Methods. 9 (1), 14 (2013).</li><li>Tsutsui, H., 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=Micrografting+device+for+testing+systemic+signaling+in+Arabidopsis.">Micrografting device for testing systemic signaling in Arabidopsis.</a> The Plant Journal. 103 (2), 918-929 (2020).</li><li>Xia, C., 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=Elucidation+of+the+mechanisms+of+long-distance+mRNA+movement+in+a+Nicotiana+benthamiana/tomato+heterograft+system.">Elucidation of the mechanisms of long-distance mRNA movement in a Nicotiana benthamiana/tomato heterograft system.</a> Plant Physiology. 177 (2), 745-758 (2018).</li><li>Li, S., 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=Unidirectional+movement+of+small+RNAs+from+shoots+to+roots+in+interspecific+heterografts.">Unidirectional movement of small RNAs from shoots to roots in interspecific heterografts.</a> Nature Plants. 7 (1), 50-59 (2021).</li><li>Ragni, L., Hardtke, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+but+thick+enough-the+Arabidopsis+hypocotyl+as+a+model+to+study+secondary+growth.">Small but thick enough-the Arabidopsis hypocotyl as a model to study secondary growth.</a> Physiologia Plantarum. 151 (2), 164-171 (2014).</li><li>Chen, I. -. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+genetics+approach+reveals+a+role+of+brassinolide+and+cellulose+synthase+in+hypocotyl+elongation+of+etiolated+Arabidopsis+seedlings.">A chemical genetics approach reveals a role of brassinolide and cellulose synthase in hypocotyl elongation of etiolated Arabidopsis seedlings.</a> Plant Science. 209, 46-57 (2013).</li><li>An, F., 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=Coordinated+regulation+of+apical+hook+development+by+gibberellins+and+ethylene+in+etiolated+Arabidopsis+seedlings.">Coordinated regulation of apical hook development by gibberellins and ethylene in etiolated Arabidopsis seedlings.</a> Cell Research. 22 (5), 915-927 (2012).</li><li>Vandenbussche, F., 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=Ethylene-induced+Arabidopsis+hypocotyl+elongation+is+dependent+on+but+not+mediated+by+gibberellins.">Ethylene-induced Arabidopsis hypocotyl elongation is dependent on but not mediated by gibberellins.</a> Journal of Experimental Botany. 58 (15-16), 4269-4281 (2007).</li><li>Vandenbussche, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Arabidopsis+mutant+alh1+illustrates+a+cross+talk+between+ethylene+and+auxin.">The Arabidopsis mutant alh1 illustrates a cross talk between ethylene and auxin.</a> Plant Physiology. 131 (3), 1228-1238 (2003).</li><li>Deslauriers, S. D., Larsen, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FERONIA+is+a+key+modulator+of+brassinosteroid+and+ethylene+responsiveness+in+arabidopsis+hypocotyls.">FERONIA is a key modulator of brassinosteroid and ethylene responsiveness in arabidopsis hypocotyls.</a> Molecular Plant. 3 (3), 626-640 (2010).</li></ol>

Summary and significance 
Formation of a graft union is crucial for successful grafting, which requires direct and undisturbed contact between the rootstock and scion. The miniature size and fragility of seedlings of small plants such as Arabidopsis makes it technically challenging to meet this requirement. One technique developed in early Arabidopsis seedling grafting methods was to insert both the scion and the rootstock into a short silicone tubing collar to support the graft ju...

Grafting is an ancient horticultural technique that became an established agricultural practice by 500 BCE1. Grafting different varieties of crop plants to improve yields was the first use of this technique, and continues to be used for this purpose today. In the past decade, grafting has attracted an increasing amount of attention as a tool for molecular biologists to study long-distance signaling in plants2,3,4,5. While grafting adult plants is relatively easy, grafting plants soon after germination is challenging. Despite this, it is sometimes required to assess the effects of long-distance signaling in processes such as plant development, environmental responses, and flowering6,7,8.
Arabidopsis thaliana has been established as the model organism in plant biology for many reasons, including its relatively small size, rendering it easy to grow inside a lab. However, the small size and fragility of Arabidopsis seedlings makes grafting young seedlings very challenging. In many cases, extensive hands-on training is required to successfully obtain seedling grafts. There have been many methodological improvements over the years that have identified ideal growing conditions and new techniques to increase the success rate of seedling grafting9,10,11. The most recent tool introduced was an Arabidopsis seedling grafting chip, that allows even inexperienced users to achieve acceptable levels of grafting success12. While this advance has significantly lowered the technical barrier of seedling grafting, the chip device is expensive, and the number of grafts that can be conducted in parallel quickly becomes cost-prohibitive.
Additionally, this device can only be used for Arabidopsis seedlings that have hypocotyl dimensions that are similar to wild-type seedlings. While Arabidopsis is the keystone species in the world of plant molecular genetics, recent work has been done in other species using seedling grafting. Examples include the grafting of soybean and the common bean, tobacco to tomato, and canola to Arabidopsis, and subsequently sampling both tissues for small RNAs13,14. Therefore, a grafting method that is accessible to most laboratories and can be easily adapted to a wide range of plant species without any major technique changes is highly desirable.
This protocol details a method that employs in-house production of a simple grafting device that allows for the full customization of grafting channel diameter and length to accommodate any seedling morphology across most plant species. The production of these devices is very affordable and highly scalable, as the only components needed are silicone elastomer, wiring or tubing of the correct size, a high precision blade, and a container to serve as a mold. Following the grafting protocol detailed here, users can achieve successful grafting rates of 45% (n = 105), comparable with previously reported grafting results10,12.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 mL conical tubes</td>
			<td>VWR International Inc</td>
			<td>10026-076</td>
			<td></td>
		</tr>
		<tr>
			<td>ACETONE (HPLC &#38; ACS Certified Solvent) 4 L</td>
			<td>VWR</td>
			<td>BJAH010-4</td>
			<td></td>
		</tr>
		<tr>
			<td>BactoAgar</td>
			<td>Sigma</td>
			<td>A1296-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>Dow SYLGARD 184 Silicone Encapsulant Clear 0.5 kg Kit</td>
			<td>Dow</td>
			<td>2646340</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Sucrose (Molecular Biology), 1 kg</td>
			<td>Fisher Scientific</td>
			<td>BP220-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Snap-Cap Microcentrifuge Flex-Tube Tubes (1.5 mL), pack of 500</td>
			<td>Fisher Scientific</td>
			<td>20901-551 / 05-402</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand High Precision #4 Style Scalpel Handle</td>
			<td>Fisher Scientific</td>
			<td>12-000-164</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Lead-Free Autoclave Tape</td>
			<td>Fisher Scientific</td>
			<td>15-901-111</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand square petri dishes</td>
			<td>Fisher Scientific</td>
			<td>FB0875711A</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica Zoom 2000 Stereo Microscope</td>
			<td>Microscope Central</td>
			<td>L-Z2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropore Tape</td>
			<td>3M</td>
			<td>B0082A9FEM</td>
			<td></td>
		</tr>
		<tr>
			<td>Murashige and Skoog Basal Medium</td>
			<td>Sigma</td>
			<td>M5519-10L</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Genesee Scientific</td>
			<td>16-101</td>
			<td></td>
		</tr>
		<tr>
			<td>potassium hydroxide</td>
			<td>VWR International Inc</td>
			<td>AA13451-36</td>
			<td></td>
		</tr>
		<tr>
			<td>Redi-earth Plug and Seedling Mix</td>
			<td>Sun Gro Horticulture</td>
			<td>SUN239274728CFLP</td>
			<td></td>
		</tr>
		<tr>
			<td>Scotts Osmocote Plus</td>
			<td>Hummert International</td>
			<td>7630600</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Design No. 22 Carbon Scalpel Blade</td>
			<td>Fisher Scientific</td>
			<td>22-079-697</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20, 500 mL</td>
			<td>Fisher Scientific</td>
			<td>BP337500</td>
			<td></td>
		</tr>
		<tr>
			<td>TWEEZER DUMONT STYL55 DUMOXEL POLS 110 MM</td>
			<td>VWR</td>
			<td>102091-580</td>
			<td></td>
		</tr>
	</tbody>
</table>

scalable, flexible, and cost-effective seedling grafting

Early-stage seedling grafting has become a popular tool in molecular genetics to study root-shoot relationships within plants. Grafting early-stage seedlings of the small model plant, Arabidopsis thaliana, is technically challenging and time consuming due to the size and fragility of its seedlings. A growing collection of published methods describe this technique with varying success rates, difficulty, and associated costs. This paper describes a simple procedure to make an in-house reusable grafting device using silicone elastomer mix, and how to use this device for seedling grafting. At the time of this publication, each reusable grafting device costs only $0.47 in consumable materials to produce. Using this method, beginners can have their first successfully grafted seedlings in less than 3 weeks from start to finish. This highly accessible procedure will allow plant molecular genetics labs to establish seedling grafting as a normal part of their experimental process. Due to the full control users have in the creation and design of these grafting devices, this technique could be easily adjusted for use in larger plants, such as tomato or tobacco, if desired.

Various aspects of the grafting strip&#39;s design were tested to identify the optimal grafting conditions that required the least amount of technical skill (Table 1). All grafting trials were completed on 0.5% sucrose MS medium, which has been previously reported to be an ideal grafting medium11,12.
Optimal seedling growth cannot be achieved with on-strip germination 
In the first iteration of th...

Watch this Scientific Journal Video about Scalable, Flexible, and Cost-Effective Seedling Grafting at JoVE.com

Scalable, Flexible, and Cost-Effective Seedling Grafting

This protocol describes a robust seedling grafting method that requires no prior experience or training and can be executed at a very low cost using materials easily accessible in most molecular biology labs.

Early-stage seedling grafting has become a popular tool in molecular genetics to study root-shoot relationships within plants. Grafting early-stage ...

Early stage seeding grafting is a powerful tool for setting long-distance signaling in plants. We hope that this method will aid in the successful adoption of seeding grafting without hands-on training. Begin by preparing the square Petri dish, measuring 100x100 millimeters for casting the silicone elastomer.Take 29 gauge wire in twist ties after removing the outer paper coating on the wire with acetone. And fully straighten the wire by rolling it on a hard, uniform surface with a heavy flat object, like a metal tube rack. Then, lay four pieces of straightened wires equidistant from one another in the square Petri dish, ensuring the wires lie flat on the bottom of the dish.Pour the mixed silicon elastomer solution on top of the wires in the Petri dish and place the cover on the dish. After allowing the silicone to cure for 24 to 48 hours at room temperature, remove the silicone sheet from the Petri dish using clean forceps and move it to a clean, flat surface. Remove the wires from the silicone sheet.To ensure the channels are open on one side, use fine-point forceps to clear the thin layer of silicone blocking the open side of the channels. Cut the silicone sheet perpendicular to the channels into three-millimeter strips using clean scissors. Place each strip in an aluminum foil envelope and seal it with autoclave tape.Autoclave the strips at 121 degrees Celsius for at least 30 minutes before storing them until required. To sterilize and vernalize the seeds, take one milliliter of 50%bleach solution containing 0.1%Tween 20 in a 1.5-milliliter microcentrifuge tube and suspend up to 100 arabidopsis seeds in it. Incubate the seed suspension at room temperature for five to 10 minutes.Post-incubation, aspirate or pipette out the bleach solution under sterile conditions. Rinse the seeds with one milliliter of sterilized distilled water. Be sure to invert the tubes adequately while rinsing the seeds.To remove any bleach solution left at the top of the tube, repeat the rinsing four times. Leave approximately 250 microliters of water with the seeds in the tube, and store the seeds in the dark at four degrees Celsius for three days. Next, before plating the seeds, place the sterile strips on the surface of the prepared MS plate to guide the seed alignment with the channels on the strips.Then, working under sterile conditions, use a 20-microliter pipette tip to aspirate and transfer the appropriate number of prepared seeds onto the plate. Once the seeds are plated, remove the sterile strips. Cover the plate.And seal one of the sides of the plate parallel to the rows of seeds with parafilm. Wrap breathable tape on top of the parafilm and around all the other edges of the plate to allow for air exchange during incubation. Carefully orient two plates vertically with their parafilm-sealed sides facing down.Separate them at the bottom by horizontally placing a 15-milliliter centrifuge tube in between, while securing them together on top with a rubber band. Ensure the plate surfaces form an angle of 100 to 110 with the benchtop surface. Store the plates in this orientation for 72 hours in total darkness at 21 degrees Celsius to allow seedling hypocotyls to grow about five millimeters in length.After which, remove the setup from the dark and keep it under a 16-hour light and eight-hour dark cycle for two to four days at the same temperature before grafting. Graft the seedlings between five and seven days after being plated. Place a grafting strip over the seedlings, fitting their hypocotyls into the channels.Gently position the seedling, so the root-hypocotyl junction remains at the bottom of the silicone strip to prepare the seedling for cutting. Working in a sterile hood with the aid of a dissection scope, prepare the scions for grafting. Using a fresh scalpel blade, make a straight, clean, perpendicular cut across the hypocotyl.Avoid pressing down and pushing the seedling into the agar. Next, remove the chute and keep the cut portion of the chute hydrated by ensuring contact with the media surface. Alternatively, the chute can be moved to a Petri dish filled with sterile distilled water.To prepare the rootstocks, carefully grasp the root within the space between the closed forceps and turn gently to pull it, leaving the cut section of the rootstocks in the middle of the strip. Then, gently pick up the desired chute using the fine-tipped forceps and insert it into the top of the the channel. Visually, confirm the contact between the scion and rootstock to obtain a successful graft.After all the grafts have been made, wrap the plates with parafilm and breathable tape, and set up the plates the same way as demonstrated before without disturbing the seedlings or silicone strips. Carefully move the plates to a growth chamber set at 26 degrees Celsius with a 16-hour light and eight-hour dark cycle. After seven to 10 days, evaluate the grafted seedlings under sterile conditions by using forceps to carefully peel off the silicone strip from one side and free the seedlings from the channels.Remove any a adventitious roots growing from the scion by cutting them with a fresh scalpel blade or crushing them with fine-tipped forceps. Once again, visually evaluate whether the rootstock has become firmly attached to the scion forming a successful graft. Move the successful grafts to seedling propagation soil and cover the soil with transparent plastic for a few days while the seedlings get established.Allow the plants to grow under the previously mentioned light and dark cycles at 21 degrees Celsius. Grafting trials to determine the optimal grafting protocol revealed an overall grafting success rate of 25%when the seeds germinated directly on the silicone strips with enclosed channels. Reorienting the seeds to keep the embryos'cotyledons and radical pointing downward during germination slightly increased the grafting efficiency to 33%Upon removing the silicone closing on the channels to allow contact between the seed and the growth medium, 85%of the seedlings germinated successfully.However, the grafting success rate remained at 31%indicating that optimal seedling growth could not be achieved with on-strip germination. Interestingly, the demonstrated protocol in which seedlings were cultivated vertically on the grafting plate before being inserted into the open channels in the strips yielded an overall grafting success rate to 48%during the first two trials. Considering the lower success rate of the third and the relatively small trial, which may be attributed to slight variations in grafting sites, the overall grafting success was found to be 45%This is comparable with the method where the seedlings were cultivated on horizontal plates, cut on a solid surface, and inserted into grafting strips.However, the demonstrated protocol is comparatively less time-consuming. Using this protocol, labs new to seedling grafting can rapidly create large numbers of grafting support devices while being able to customize the device to fit their specific needs easily.

scalable-flexible-and-cost-effective-seedling-grafting

Research

JoVE Journal

Biology

BioMEMS and Cellular Biology: Perspectives and Applications

The ability to culture cells has revolutionized hypothesis testing in basic cell and molecular biology research. It has become a standard methodology in drug screening, toxicology, and clinical assays, and is increasingly used in regenerative medicine. However, the traditional cell culture methodology   essentially consisting of the immersion of a large population of cells in a homogeneous fluid medium and on a homogeneous flat substrate   has become increasingly limiting both from a fundamental and practical perspective. Microfabrication technologies have enabled researchers to design, with micrometer control, the biochemical composition and topology of the substrate, and the medium composition, as well as the neighboring cell type in the surrounding cellular microenvironment. Additionally, microtechnology is conceptually well-suited for the development of fast, low-cost in vitro systems that allow for high-throughput culturing and analysis of cells under large numbers of conditions. In this interview, Albert Folch explains these limitations, how they can be overcome with soft lithography and microfluidics, and describes some relevant examples of research in his lab and future directions.

The ability to culture cells has revolutionized hypothesis testing in basic cell and molecular biology research. It has become a standard methodology in ...

Combining Peripheral Nerve Grafting and Matrix Modulation to Repair the Injured Rat Spinal Cord

Traumatic injury to the spinal cord (SCI) causes death of neurons, disruption of motor and sensory nerve fiber (axon) pathways and disruption of communication with the brain. One of the goals of our research is to promote axon regeneration to restore connectivity across the lesion site. To accomplish this we developed a peripheral nerve (PN) grafting technique where segments of sciatic nerve are either placed directly between the damaged ends of the spinal cord or are used to form a bridge across the lesion. There are several advantages to this approach compared to transplantation of other neural tissues; regenerating axons can be directed towards a specific target area, the number and source of regenerating axons is easily determined by tracing techniques, the graft can be used for electrophysiological experiments to measure functional recovery associated with axons in the graft, and it is possible to use an autologous nerve to reduce the possibility of graft rejection. In our lab we have performed both autologous (donor and recipient are the same animal) and heterologous (donor and recipient are different animals) grafts with comparable results. This approach has been used successfully in both acute and chronic injury situations. Regenerated axons that reach the distal end of the PN graft often fail to extend back into the spinal cord, so we use microinjections of chondroitinase to degrade inhibitory molecules associated with the scar tissue surrounding the area of SCI. At the same time we have found that providing exogenous growth and trophic molecules encourages longer distance axonal regrowth into the spinal cord. Several months after transplantation we perform a variety of anatomical, behavioral and electrophysiological tests to evaluate the recovery of function in our spinal cord injured animals. This experimental approach has been used successfully in several spinal cord injury models, at different levels of injury and in different species (mouse, rat and cat). Importantly, the peripheral nerve grafting approach is effective in promoting regeneration by acute and chronically injured neurons.

Traumatic injury to the spinal cord disrupts communication with the brain. To restore lost connectivity we utilize a peripheral nerve graft to provide a substratum for regenerating fibers in combination with neurotrophic factors and matrix-modulating enzymes to remove inhibitory molecules to promote long distance growth.

Traumatic injury to the spinal cord (SCI) causes death of neurons, disruption of motor and sensory nerve fiber (axon) pathways and disruption of ...

Diagnostic Necropsy and Selected Tissue and Sample Collection in Rats and Mice

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal colony. Proper collection of tissues for further histological processing can impact the quality of testing results. This article describes the conduct of a basic gross examination including identification of heart, liver, lungs, kidneys, and spleen, as well as how to collect those organs. Additionally four of the more difficult tissue/sample collection techniques are demonstrated. Lung collection and perfusion can be particularly challenging as the tissue needs to be properly inflated with a fixative in order for inside of the tissue to fix properly and to enable thorough histologic evaluation. This article demonstrates the step by step technique to remove the lung and inflate it with fixative in order to achieve optimal fixation of the tissue within 24 hours. Brain collection can be similarly challenging as the tissue is soft and easily damaged. This article demonstrates the step by step technique to expose and remove the brain from the skull with minimal damage to the tissue. The mesenteric lymph node is a good sample type in which to detect many common infectious agents as enteric viruses persist longer in the lymph node than they are shed in feces. This article demonstrates the step by step procedure for locating and aseptically removing the mesenteric lymph node. Finally, identification of infectious agents of the respiratory tract may be performed by bacterial culture or PCR testing of nasal and/or bronchial fluid aspirates taken at necropsy. This procedure describes obtaining and preparing the respiratory aspirate sample for bacterial culture and PCR testing.

This article describes the procedures for conducting a basic postmortem examination of a mouse or rat, and the collection of basic organs, as well as more challenging sample types from for histological, microbiological, and PCR evaluation.

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal ...

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation.
Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

The extracellular matrix plays a major role in defining the microenvironment of cells and in modulating cell behavior and phenotype. We describe a rapid method for the isolation of cell-derived extracellular matrix, which can be adapted to different scales for microscopic, biochemical, proteomic, or functional studies.

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and ...

An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. Three-dimensional (3D) cell culture techniques are a recent innovation developed to address the shortcomings of adherent cell culture. While several techniques for generating tissue analogues in vitro have been developed, these methods are frequently complex, expensive to establish, require specialized equipment, and are generally limited by compatibility with only certain cell types. Here, we describe a rapid and flexible protocol for aggregating cells into multicellular 3D spheroids of consistent size that is compatible with growth of a variety of tumor and normal cell lines. We utilize varying concentrations of serum and methyl cellulose (MC) to promote anchorage-independent spheroid generation and prevent the formation of cell monolayers in a highly reproducible manner. Optimal conditions for individual cell lines can be achieved by adjusting MC or serum concentrations in the spheroid formation medium. The 3D spheroids generated can be collected for use in a wide range of applications, including cell signaling or gene expression studies, candidate drug screening, or in the study of cellular processes such as tumor cell invasion and migration. The protocol is also readily adapted to generate clonal spheroids from single cells, and can be adapted to assess anchorage-independent growth and anoikis-resistance. Overall, our protocol provides an easily modifiable method for generating and utilizing 3D cell spheroids in order to recapitulate the 3D microenvironment of tissues and model the in vivo growth of normal and tumor cells.

Here, we describe a rapid and flexible protocol for the formation of 3D cell spheroids through cell aggregation. This is easily adapted to multiple cell types and is suitable for use in a variety of applications including cell migration, invasion, or anoikis assays, and for imaging and quantifying cell-matrix interactions.

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. ...

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR)

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In circadian rhythms research, for example, clock gene fusions with firefly luciferase give rise to robust rhythms in cellular bioluminescence that persist over many days. Technical limitations associated with photomultiplier tubes (PMT) or conventional microscopy-based methods for bioluminescence quantification have typically demanded that cells and tissues be maintained under quite non-physiological conditions during recording, with a trade-off between sensitivity and throughput. Here, we report a refinement of prior methods that allows long-term bioluminescence imaging with high sensitivity and throughput which supports a broad range of culture conditions, including variable gas and humidity control, and that accepts many different tissue culture plates and dishes. This automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) also allows the observation of spatial variations in luciferase expression across a cell monolayer or tissue, which cannot readily be observed by traditional methods. We highlight how the ALLIGATOR provides vastly increased flexibility for the detection of luciferase activity when compared with existing methods.

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR

Genetically encoded luciferase is a popular non-invasive reporter of gene expression. Use of an automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) enables longitudinal recording from bioluminescent cells under a wide range of conditions. Here we show how ALLIGATOR may be used in the context of circadian rhythms research.

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In ...

A Cost Effective and Adaptable Scratch Migration Assay

Cell migration is a key component in both physiological and pathological events. Normal cell migration is required for essential functions such as development and mounting an immune response. When a defect or alteration occurs with the cell migration process, it can have detrimental outcomes (i.e., cancer metastasis, wound healing, and scar formation). Due to the importance of cell migration, it is necessary to have access to a cell migration assay that is affordable, adaptable, and repeatable. Utilizing the common scratch migration assay, we have developed a new approach to analyzing cell migration that uses general laboratory equipment. The method described uses visual markers that allow for recapturing specific areas of interest without the use of time-lapse microscopy. In addition, it provides flexibility in the experimental design, ranging from altering the migration matrix substrate to the addition of pharmacological modifiers. Furthermore, this protocol outlines a way to account for the area of cell migration, which is not considered by several methods when examining cell migration. This new approach offers a scratch migration assay to a larger audience and will provide greater opportunity for researchers to examine the physiological and pathophysiological impact of cell migration.

We present a cost-effective method to the scratch migration assay that provides a new approach for determining cell migration without the use of equipment-intensive methods. While fibroblasts were used in this protocol, it can be adapted and utilized to study additional cell types and influences on cell migration.

Cell migration is a key component in both physiological and pathological events. Normal cell migration is required for essential functions such as ...

An Easy and Flexible Inoculation Method for Accurately Assessing Powdery Mildew-Infection Phenotypes of Arabidopsis and Other Plants

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in turn requires accurate determination of disease phenotypes of plants upon infection with a particular fungal pathogen. However, accurate disease phenotyping with unculturable biotrophic fungal pathogens such as powdery mildew is not easy to achieve and can be a rate-limiting step of a research project. Here, we have developed a safe, efficient, and easy-to-operate disease phenotyping system using the Arabidopsis-powdery mildew interaction as an example. This system mainly consists of three components: (i) a wooden inoculation box fitted with a removable lid mounted with a stainless steel or nylon mesh of ~50 &#181;m pores for inoculating a flat of plants with fungal spores, (ii) a transparent plastic chamber with a small front opening for minimizing spore escape while conducting inoculation inside, and (iii) a spore-dislodging and distribution method for even and effective inoculation. The protocols described here include the steps and parameters for making the inoculation box and the plastic chamber at a low cost, and a video demonstration of how to use the system to enable even inoculation with powdery mildew spores, thereby improving accuracy and reproducibility of disease phenotyping.

We present a protocol for constructing a simple spore-distribution system consisting of an inoculation box with a ~50 &#181;m mesh and a transparent plastic chamber. This can be used to evenly inoculate plants with powdery mildew spores, thereby enabling accurate and reproducible assessment of disease phenotypes of plants under study.

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in ...

High-throughput, Robust and Highly Time-flexible Method for Surface Sterilization of Arabidopsis Seeds

Arabidopsis is by far the plant model species most widely used for functional studies. The surface sterilization of Arabidopsis seeds is a fundamental step required towards this end. Thus, it is paramount to establish high-throughput Arabidopsis seed surface sterilization methods to handle tens to hundreds of samples (e.g., transgenic lines, ecotypes, or mutants) at once. A seed surface sterilization method based on the efficient elimination of liquid in tubes with a homemade suction device constructed from a common vacuum pump is presented in this study. By dramatically reducing labor-intensive hands-on time with this method handling several hundreds of samples in one day is possible with little effort. Series time-course analyses further indicated a highly flexible time range of surface sterilization by maintaining high germination rates. This method could be easily adapted for surface sterilization of other kinds of small seeds with simple customization of the suction device according to the seed size, and the speed desired to eliminate the liquid.

A high-throughput protocol for the surface sterilization of Arabidopsisthaliana (Arabidopsis) seeds is provided, optimizing the liquid handling steps with a simple suction device constructed with a vacuum pump. Hundreds of seed samples can be surface-sterilized in one day.

Arabidopsis is by far the plant model species most widely used for functional studies. The surface sterilization of Arabidopsis seeds is a fundamental ...